Anti-nme antibody and method of treating cancer or cancer metastasis

ABSTRACT

The present application discloses anti-NME antibodies and their use in treating or preventing diseases.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 15, 2023, isnamed 56699-743_831_SL.txt and is 465,564 bytes in size.

BACKGROUND OF THE INVENTION Field of the Invention

The present application relates to NME proteins, peptides derived fromNME proteins, and antibodies generated from the peptides thereof orantibody or antibody fragments selected by virtue of their ability tobind to said peptides. The present application also relates to treatingor preventing diseases associated with the expression of NME in apatient.

2. General Background and State of the Art

NDPK (nucleoside diphosphate protein kinase) proteins are a family ofproteins grouped together because they all contain an NDPK domain. Thefirst NME proteins discovered, previously called NM23 proteins, wereNM23-H1 and NM23-H2. For decades it was unclear whether they induceddifferentiation or prevented differentiation of hematopoietic cells. Theinventors previously discovered that NM23-H1 prevents differentiationwhen it is a dimer, which binds to the MUC1* growth factor receptor, butat higher concentrations NM23-H1 becomes a hexamer, which does not bindto MUC1*, and it induces differentiation. NM23 used to be called ametastasis suppressor when it was found that it was under-expressed insome very aggressive cancers. The present inventors previously disclosedthat NM23-H1 dimers bind to and dimerize the extracellular domain of theMUC1* growth factor receptor that is over expressed on the vast majorityof cancers and such binding promotes the growth of cancer cells.Conversely, at higher concentrations, NM23 forms tetramers and hexamersthat do not bind to MUC1* and do not promote tumorigenesis. Veryrecently more NME family proteins (NME 1-10) have been discoveredalthough until now, their functions have not been elucidated. NME7 is anewly discovered NME family protein, but its NDPK domain has noenzymatic activity, unlike other NME family members. NME7 is either notexpressed at all in adult tissues or is expressed at extremely lowlevels.

SUMMARY OF THE INVENTION

The present application is directed to a method of treating orpreventing cancer in a subject, comprising administering to the subjectan antibody made against a member of the NME family. The NME family maybe NME7 family. The antibody may bind to NME7. The antibody may bind toNME7AB or NME7AB-like protein. The antibody may bind to NME7-X1. Theantibody may inhibit binding between NME7 and its cognate bindingpartner. The cognate binding partner may be MUC1*. The cognate bindingpartner may be PSMGFR portion of the MUC1* extracellular domain. In oneaspect, the antibody may be generated or selected for its ability tobind to a peptide selected from those listed in FIGS. 6-9 (SEQ ID NOS:88to 145). Preferably, the peptide may be selected from those listed inFIG. 9 (SEQ ID NOS:141 to 145).

The peptide may be highly homologous to, or to which is added orsubtracted up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or upto 1 amino acid residues at the N-terminus or C-terminus, of thepeptides listed in FIG. 6-9 (SEQ ID NOS:88 to 145). In one aspect, theantibody may be selected for its ability to bind to NME7AB or NME7-X1but not to NME1. The antibody may be polyclonal, monoclonal, bivalent,monovalent, bispecific, an antibody fragment containing the variableregion, or an antibody mimic. The antibody may be human or humanized.The antibody may be a single chain scFv.

In another aspect, the invention is directed to a method of treating orpreventing cancer in a subject, comprising administering to the subjecta peptide that is highly homologous or identical to regions of NME7AB.The peptide may be at least 80% homologous to one or more of thepeptides listed in FIG. 6 . The peptide may be at least 80% homologousto one or more of the peptides listed in FIG. 7 . The peptide may be atleast 80% homologous to one or more of the peptides listed in FIG. 8 .The peptide may be at least 80% homologous to one or more of thepeptides listed in FIG. 9 . The peptide may be selected from peptideslisted in FIG. 6-9 (SEQ ID NOS:88 to 145). The peptide may be selectedfrom those listed in FIG. 9 (SEQ ID NOS:141 to 145). Or, the peptide maybe highly homologous to, or to which is added or subtracted up to 7, upto 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid residuesat the N-terminus or C-terminus, of the peptides listed in FIG. 6-9 (SEQID NOS:88 to 145). The peptide may be connected to another peptide via aspacer or linker.

In another aspect, the invention is directed to a chimeric antigenreceptor (CAR), for the treatment or prevention of cancer wherein thetargeting extracellular portion of the CAR comprises at least a peptidefragment of a member of the NME family. NME family may be NME7 family.The member of the NME7 family may be NME7. Or, the member of the NME7family may be NME7AB or NME7AB-like protein. The member of the NME7family may be also NME7-X1. The targeting extracellular portion of theCAR may include a peptide of the peptides listed in FIG. 6-9 (SEQ IDNOS:88 to 145). The peptide may be selected from those listed in FIG. 9(SEQ ID NOS:141 to 145). The peptide may include a peptide, which ishighly homologous to, or to which is added or subtracted up to 7, up to6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid residues atthe N-terminus or C-terminus, of the peptides listed in FIG. 6-9 (SEQ IDNOS:88 to 145). The peptide may be connected to another peptide via aspacer or linker.

In yet another aspect, the invention is directed to a method of treatingor preventing cancer or cancer metastasis, comprising engineering thechimeric antigen receptor according to claim 3, into an immune systemcell and administering the cell to a subject in need thereof.

In another aspect, the invention is directed to a chimeric antigenreceptor (CAR), for the treatment or prevention of cancer, wherein thetargeting extracellular portion of the chimeric antigen receptorcomprises a portion of an antibody that binds to NME7AB, NME7AB-likeprotein or NME7-X1. The portion of the antibody may be a single chainscFv or may be human or humanized.

In yet another aspect, the invention is directed to a method ofvaccinating a person against cancer or metastatic cancer comprisingimmunizing the person with a peptide fragment of a member of the NMEfamily. The NME family may be NME7 family. The member of the NME7 familymay be NME7 or NME7b. The member of the NME7 family may be NME7AB orNME7AB-like protein. The NME7 family may be NME7-X1. The immunizingpeptide may be a peptide from the peptides listed in FIG. 6-9 (SEQ IDNOS:88 to 145). Preferably, the peptide may be selected from thoselisted in FIG. 9 (SEQ ID NOS:141 to 145). The immunizing peptide mayinclude a peptide, which is highly homologous to, or to which is addedor subtracted up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, orup to 1 amino acid residues at the N-terminus or C-terminus, of thepeptides listed in FIG. 6-9 (SEQ ID NOS:88 to 145). The immunizingpeptide may be connected to another peptide via a spacer or linker.

In yet another aspect, the invention is directed to a method of treatingor preventing cancer in a subject, comprising administering to thesubject a nucleic acid that inhibits the expression of NME7, NME7b,NME7AB-like protein or NME7-X1. The nucleic acid may be an anti-sensenucleic acid that suppresses expression of NME7, NME7AB-like protein orNME7-X1. The nucleic acid may be an inhibitory RNA, siRNA, RNAi, orshRNA that inhibits expression of NME7, NME7AB-like protein or NME7-X1.

In another aspect, the invention is directed to a method of treating orpreventing cancer in a subject, comprising administering to the subjectgenetically edited nucleic acids that inhibit expression of NME7, NME7b,NME7AB-like protein or NME7-X1. The genetically edited nucleic acidsthat inhibit expression of NME7, NME7b, NME7AB-like protein or NME7-X1may be inserted into cells that may be then administered to the patient.The genetically edited nucleic acids that inhibit expression of NME7,NME7b, NME7AB-like protein or NME7-X1 may be inserted into cells using aviral vector. The viral vector may be a lentiviral system.

In another aspect, the invention is directed to a method of growingcancer cells comprising contacting the cells with NME7AB, NME7b,NME7AB-like protein or NME7-X1, 2i or 5i. The method may includeculturing the cells in a medium that contains NME7AB, NME7b, NME7AB-likeprotein or NME7-X1, 2i or 5i, or growing cells in an animal thatexpresses human NME7AB, NME7b, NME7AB-like protein or NME7-X1, or towhich NME7AB, NME7b, NME7AB-like protein or NME7-X1 is administered. Thecancer cells may be breast, prostate, ovarian, colorectal, pancreatic,liver, melanoma or brain cancer cells. Drug candidates may be tested onthe cells. The efficacy of the drugs may be assessed by comparing cancergrowth to a no drug control or comparing expression levels of metastaticmarkers or stem cell markers to a no drug control or comparing theability of the resultant cells to form tumors in animals from low cellcopy number compared to a no drug control and determining the efficacyof a candidate drug for the treatment of cancer or metastasis. The cellsmay be obtained from a patient being assessed for treatment for cancerand drugs that would be effective for that patient are selected based onresults using methods described above. The cells may not be obtainedfrom a patient being assessed for treatment for cancer but drugs thatwould be effective for that patient are selected based on results usingthe methods described above.

In another aspect, the invention is directed to a method of generatingantibodies or antibody-like molecules from peptides or peptide mimicshaving a sequence derived from the sequence of NME. The NME may be NME7.The peptide may be used as an immunogen to generate antibodies orantibody-like molecules. The peptide may be administered to an animal togenerate anti-NME7 antibodies. The peptide may be administered to ahuman to generate anti-NME7 antibodies. The peptide may have a sequencelisted in FIG. 6-9 (SEQ ID NOS:88 to 145). Preferably, the peptide maybe selected from those listed in FIG. 9 (SEQ ID NOS:141 to 145). Thepeptide may include a peptide, which is highly homologous to, or towhich is added or subtracted up to 7, up to 6, up to 5, up to 4, up to3, up to 2, or up to 1 amino acid residues at the N-terminus orC-terminus, of the peptides listed in FIG. 6-9 (SEQ ID NOS:88 to 145).

In another aspect, the invention is directed to a method of detectingpresence of cancer or the progression of cancer, comprising the stepsof:

-   -   1) obtaining a sample from a patient having cancer or at risk of        developing a cancer;    -   2) subjecting that sample to an assay capable of detecting or        measuring levels of a member of the NME7 family, or levels of        nucleic acids encoding a member of the NME7 family;    -   3) comparing levels of the measured member of the NME7 family or        the member of the NME7 family-encoding nucleic acids in the test        sample to levels in control patients or control cells;    -   4) determining that the levels of the member of the NME7 family        or nucleic acids encoding the member of the NME7 family are        elevated compared to the controls; and    -   5) concluding that the donor of the test sample has cancer or        has had a progression of cancer if the control to which the test        was compared came from a donor previously diagnosed with a        cancer. In this method, the detection of the member of the NME7        family in circulation or in a tissue may be an indicator of        cancer in a patient. The member of the NME7 family may be NME7,        NME7b, NME7-X1, or NME7AB-like protein.

In yet another aspect, the invention is directed to a method comprising:

-   -   detecting presence of a member of the NME7 family or MUC1* in a        patient; and    -   administering anti-NME7 or anti-MUC1* antibody or antibodies to        the patient exhibiting the member of the NME7 family or MUC1*        expression. The member of the NME7 family may be NME7, NME7b,        NME7-X1, or NME7AB-like protein.    -   In yet another aspect, the invention is directed to a method for        treating or preventing cancer comprising:    -   1) obtaining a sample from a patient suspected of having a        cancer or at risk of developing a cancer or at risk of        developing a metastatic cancer;    -   2) measuring an amount of the member of an NME7 family or a        member of the NME7 family encoding nucleic acid, wherein the        measured levels are significantly above those measured in a        control sample;    -   3) determining that the patient has a cancer or has developed a        more aggressive or a metastatic cancer;    -   4) administering to the patient an effective amount of a        therapeutic agent that suppresses expression of the member of        the NME7 family, inhibits cleavage of NME7 or inhibits NME7        binding to its targets. The target of the member of the NME7        family may be MUC1*. The target of the member of the NME7 family        may be PSMGFR portion of the MUC1* extracellular domain. The        member of the NME7 family may be NME7, NME7b, NME7-X1, or        NME7AB-like protein.

In any of the methods above regarding cancer, cancer may include breast,prostate, ovarian, colorectal, pancreatic, liver, melanoma or braincancer.

In one aspect, the invention is directed to an NME7 specific antibody orfragment thereof that binds to the NME7 B3 peptide of SEQ ID NO:145 orSEQ ID NO:169. The antibody may be monoclonal antibody or bivalent,monovalent, an Fab, or a single chain variable fragment antibody (scFv).The antibody may be linked to an antibody drug conjugate. The drug maybe linked to a toxin or pro-toxin.

The invention is also directed to an isolated nucleic acid encoding theantibody.

The invention is also directed to an isolated hybridoma expressing themonoclonal antibody discussed above. The antibody may specifically bindto NME7AB or NME7-X1, but not to NME1. The antibody may disruptinteraction between NME7AB and MUC1* extra cellular domain or betweenNME7-X1 and MUC1* extra cellular domain. Or, the antibody may disruptbinding between NME7AB and PSMGFR or between NME7-X1 and PSMGFR.Further, the antibody may disrupt binding between NME7AB and N-10 orbetween NME7-X1 and N-10.

In another aspect, the antibody may not disrupt interaction betweenNME7AB and MUC1* extra cellular domain or between NME7-X1 and MUC1*extra cellular domain. NME7_(AB) or NME7-X1 may bind to the N-10 peptide(SEQ ID NO:170) but not to a C-10 peptide (SEQ ID NO:171). Inparticular, the antibody may be 5A1, 4A3, 5D4, or 4P3.

The antibody may comprise an amino acid sequence in the heavy chainvariable region comprising the following:

-   -   in the CDR1 region YTFTNYGMN (SEQ ID NO:439);    -   in the CDR2 region WINTYTGEPTYVDDFKG (SEQ ID NO:440); and    -   in the CDR3 region LRGIRPGPLAY (SEQ ID NO:441); and    -   an amino acid sequence in the light chain variable region        comprising the following:    -   in the CDR1 region SASSSVSYMN (SEQ ID NO:444);    -   in the CDR2 region GISNLAS (SEQ ID NO:445); and    -   in the CDR3 region QQRSSYPPT (SEQ ID NO:446).

An example of such an antibody above is 5A1.

In another aspect, the antibody may comprise an amino acid sequence inthe heavy chain variable region comprising the following:

-   -   in the CDR1 region NTFTEYTMH (SEQ ID NO:429);    -   in the CDR2 region GFNPNNGVTNYNQKFKG (SEQ ID NO:430); and    -   in the CDR3 region RYYHSTYVFYFDS (SEQ ID NO:431); and    -   an amino acid sequence in the light chain variable region        comprising the following:    -   in the CDR1 region SASQGISNYLN (SEQ ID NO:434);    -   in the CDR2 region YTSSLHS (SEQ ID NO:435); and    -   in the CDR3 region QQYSKLPYT (SEQ ID NO:436).

An example of such an antibody above is 5D4.

In another aspect, the antibody may comprise an amino acid sequence inthe heavy chain variable region comprising the following:

-   -   in the CDR1 region NTFTEYTMH (SEQ ID NO:388);    -   in the CDR2 region GFNPNNGVTNYNQKFKG (SEQ ID NO:389); and    -   in the CDR3 region RYYHSLYVFYFDY (SEQ ID NO:390); and    -   an amino acid sequence in the light chain variable region        comprising the following:    -   in the CDR1 region SASQGISNYLN (SEQ ID NO:393);    -   in the CDR2 region YTSSLHS (SEQ ID NO:394); and    -   in the CDR3 region QQYSKLPYT (SEQ ID NO:395).

An example of such an antibody above is 4A3.

In another aspect, the antibody may comprise an amino acid sequence inthe heavy chain variable region comprising the following:

-   -   in the CDR1 region NTFTEYTMH (SEQ ID NO:388);    -   in the CDR2 region GFNPNNGVTNYNQKFKG (SEQ ID NO:389); and    -   in the CDR3 region RYYHSLYVFYFDY (SEQ ID NO:390); and    -   an amino acid sequence in the light chain variable region        comprising the following:    -   in the CDR1 region ITSTDIDDDMN (SEQ ID NO: 1144);    -   in the CDR2 region EGNTLRP (SEQ ID NO: 1145); and    -   in the CDR3 region LQSDNLPLT (SEQ ID NO: 1146).

An example of such an antibody above is 4P3.

The antibody may be human, humanized or an engineered antibody mimic.

The antibody may be non-human, such as murine or camelid.

The invention is also directed to a method of administering to a patientfor prevention or treatment of cancer comprising administering to thepatient a composition comprising the antibody described above.

The invention is also directed to a method for preventing or treatingcancer metastasis in a patient, comprising administering to the patienta composition comprising the antibody described above.

The invention is also directed to a method for diagnosing cancer orcancer metastasis comprising contacting a patient specimen and normalspecimen with the antibody above, and comparing the results from bothspecimen, wherein presence of positive binding to the antibody in thepatient specimen indicates the presence of cancer or cancer metastasisin the patient. The antibody may be linked to an imaging agent. Thepatient specimen may be blood, bodily fluid, tissue, circulating cells,in vitro, in vivo, including intra-operative.

The invention is also directed to a cell that is engineered to expressan anti-NME7_(AB) antibody or fragment thereof. The cell may be animmune cell, such as T cell or NK cell, or a stem or progenitor cell,preferably stem or progenitor cell that is then differentiated to becomea T cell.

The cell may comprise a chimeric antigen receptor (CAR) that recognizestumor associated antigen. Expression of the anti-NME7 antibody may beinducible. Nucleic acid encoding an anti-NME7_(AB) antibody may beinserted into the Foxp3 enhancer or promoter. The anti-NME7_(AB)antibody may be in an NFAT-inducible system. NFATc1 response element maybe inserted upstream of the antibody sequence that is inserted intoFoxp3 enhancer or promoter region.

The anti-NME7_(AB) antibody or fragment thereof may bind to the NME7 B3peptide, or disrupt binding of NME7_(AB) or NME7-X1 to the PSMGFRpeptide of the MUC1* extra cellular domain.

The CAR may recognize a tumor associated antigen and an anti-NME7antibody. The tumor associated antigen may be MUC1*.

The invention is also directed to an anti-cancer vaccine comprising acomposition comprising one or more peptides derived from NME7_(AB)listed in FIG. 6 -FIG. 9 or a peptide having at least 80%, 85%, 90%,95%, 97% sequence identity thereof as the immunogenicity elicitingportion. The peptide may be a peptide of SEQ ID NOS:141-145 or a peptidehaving at least 80%, 85%, 90%, 95%, 97% sequence identity thereof. Thepeptide may be a peptide of SEQ ID NO: 145 or a peptide having at least80%, 85%, 90%, 95%, 97% sequence identity thereof.

In another aspect, the invention is directed to a BiTE comprising theabove-described antibody.

In yet another aspect, the invention is directed to a method ofgenerating anti-NME7_(AB) antibodies wherein Cysteine residue in theNME7 B3 peptide is mutated to avoid disulfide bonding.

In yet another aspect, the invention is directed to a method ofgenerating cells with enhanced metastatic potential comprising culturingthe cells with NME7_(AB) or NME7-X1.

The invention is also directed to a cell that is engineered to expressNME7_(AB) or NME7-X1, a transgenic animal that expresses NME7_(AB) orNME7-X1, wherein the NME7_(AB) or NME7-X1 may be human, and also whereinexpression of NME7_(AB) or NME7-X1 may be inducible.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

The present invention will become more fully understood from thedetailed description given herein below, and the accompanying drawingswhich are given by way of illustration only, and thus are not limitativeof the present invention, and wherein;

FIG. 1 shows a graph of HRP signal from ELISA sandwich assay showingNME7-AB dimerizes MUC1* extra cellular domain peptide.

FIG. 2 is a graph of RT-PCR measurements of gene expression for stemcell markers and cancer stem cell markers for T47D cancer cells afterbeing cultured in traditional media or a media containing NME7, whereincells that became non-adherent (floaters) were analyzed separate fromthose that remained adherent.

FIG. 3 is a graph of RT-PCR measurements of gene expression for avariety of stem and putative cancer stem cell markers for DU145 prostatecancer cells. Cells were cultured either in traditional media or a mediacontaining NME1 dimers (“NM23”) or NME7 (NME7-AB). Rho kinase inhibitorwas not used because by passage 2, cells remained adherent.

FIG. 4 is a graph of RT-PCR measurement of the metastatic markers andpluripotent stem cell markers showing that the 2i inhibitors (GSK3-betaand MEK inhibitors), which were previously shown to revert stem cells toa more naïve state, also induce cancer cells to a more metastatic statealthough not as well as NME7_(AB).

FIG. 5 is a sequence alignment between human NME1 and human NME7-A or -Bdomain. Figure discloses SEQ ID NOS 1137, 1140, 1137 and 1143,respectively, in order of appearance.

FIG. 6 lists immunogenic peptides from human NME7 with low sequenceidentity to NME1 and selected for their ability to generate therapeuticanti-NME7 antibodies for the treatment or prevention of cancers. Figurediscloses SEQ ID NOS 88-108, 91, and 110-118, respectively, in order ofappearance.

FIG. 7 lists immunogenic peptides from human NME7 that may be importantfor structural integrity or for binding to MUC1* selected for theirability to generate therapeutic anti-NME7 antibodies for the treatmentor prevention of cancers. Figure discloses SEQ ID NOS 122, 114, 124-129,128, and 131-133, respectively, in order of appearance.

FIG. 8 lists immunogenic peptides from human NME1 that may be importantfor structural integrity or for binding to MUC1* and selected for theirability to generate therapeutic anti-NME7 antibodies for the treatmentor prevention of cancers. Figure discloses SEQ ID NOS 134-140,respectively, in order of appearance.

FIG. 9 lists immunogenic peptides from human NME7 selected for their lowsequence identity to NME1 and for their homology to bacterial NME1proteins that have been implicated in cancers. These peptides arepreferred for their ability to generate therapeutic anti-NME7 antibodiesfor the treatment or prevention of cancers. The peptides shown in thisFigure include and added Cysteine covalently bound at the C-terminalend. Figure discloses SEQ ID NOS 107, 111, 143-145, and 169,respectively, in order of appearance.

FIGS. 10A-10B show graphs of ELISA assays in which either NME7-AB (FIG.10A) or NME1 (FIG. 10B) is adsorbed to the plate and anti-NME7antibodies generated by NME7 peptides A1, A2, B1, B2 and B3 are testedfor their ability to bind to NME7 but not to NME1. C20 is an anti-NME1antibody.

FIG. 11 shows graphs of ELISA assays in which anti-NME7 antibodiesgenerated are tested for their ability to inhibit binding of NME7-AB toa surface immobilized MUC1* peptide but not inhibit binding of NME1.

FIG. 12 shows a graph of a cancer cell growth experiment in which breastcancer cells were grown in the presence or absence of NME7 antibodies orshort peptides derived from NME7, which were used to generate or selectthe antibodies. In addition, an antibody generated by immunization withnearly the entire NME7-AB peptide, amino acids 100-376, was shown toinhibit cancer cell growth.

FIG. 13 shows a graph of a cancer cell growth experiment in which breastcancer cells were grown in the presence or absence of combinations ofNME7 antibodies or combinations of the short peptides derived from NME7,which were used to generate or select the antibodies. Both antibodies aswell as their immunizing NME7-AB peptides inhibited growth of cancercells.

FIGS. 14A-14B show tables of scientist observations when cancer cellswere grown in either NME7-AB or 2i inhibitors, which both are able totransform cancer cells to a more metastatic state, and in the presenceor absence of NME7 derived peptides A1, A2, B1, B2 and B3. The NME7-ABpeptides inhibited the transition of adherent cancer cells to thefloater cells, which RT-PCR measurements show have increased expressionof metastatic markers, especially CXCR4.

FIGS. 15A-15C show graphs of RT-PCR measurements of expression of CXCR4and other metastatic markers in T47D breast cancer cells that were grownin either NME7-AB or 2i inhibitors, each of which transform cancer cellsto a more metastatic state, and the inhibitory effect of anti-NME7antibodies on the metastatic transformation. FIG. 15A shows PCR graph ofCXCR4 expression of T47D cancer cells grown in NME7_(AB) or 2i in thepresence or absence of anti-NME7 antibodies. FIG. 15B shows a graph ofRT-PCR measurements of CXCR4, CHD1 and SOX2 expression in T47D breastcancer cells that were grown in 2i inhibitors for 72 hours or 144 hours,in the presence of NME7_(AB) immunizing peptides and shows the peptidesare themselves inhibitory to the metastatic transformation. Peptides A1,A2 and B1 which were used in the inhibitory Combo 2 and 3 in FIG. 15Aare also inhibitory as peptides. Peptide B3 is the most inhibitory andis the immunizing peptide for antibody 61 which was the most inhibitoryantibody tested in FIG. 15A. FIG. 15C shows the graph of FIG. 15B withthe scale of the Y-axis reduced.

FIG. 16 shows a table of recorded RNA levels in samples that were usedfor RT-PCR measurement of CXCR4 in FIG. 31 as well as the thresholdcycle number for CXCR4 expression as well as for the controlhousekeeping gene.

FIG. 17 shows a graph of RT-PCR measurement of the expression of NME7-X1in a panel of human stem cells and cancer cells.

FIG. 18 shows a graph of RT-PCR measurement of the expression of NME7,NME7a, NME7b and NME7-X1 in a panel of human stem cells and cancercells. NME7a is full-length NME7, NME7b is missing a small portion ofthe DM10 domain, NME7-X1 is missing all of the DM10 domain and a smallportion of the N-terminus of the first NDPK A domain. The bar labeledNME7 means that primers were used that detected both NME7a and NME7b.

FIGS. 19A-19F show photographs of Western blots in which various cancercell lines are probed for expression of NME7 species using antibodiesgenerated by immunization with NME7 derived peptides. FIG. 19A showsWestern blot wherein antibody 52 that binds to the A1 peptide is used toprobe a panel of cells for the presence of full-length NME7, NME7_(AB)or NME7-X1. FIG. 19B shows Western blot wherein antibody 56 that bindsto the B1 peptide is used to probe a panel of cells for the presence offull-length NME7, NME7_(AB) or NME7-X1.

FIG. 19C shows Western blot wherein antibody 61 that binds to the B3peptide is used to probe a panel of cells for the presence offull-length NME7, NME7_(AB) or NME7-X1. FIG. 19D shows Western blotwherein commercially available polyclonal antibody H278, raised againstboth the NME7 A and B domain, is used to probe a panel of cells for thepresence of NME7. As the figure shows, antibody H278 also recognizesNME1. FIG. 19E shows a gel published on website for commerciallyavailable anti-NME7 antibody B9, showing it binds to a species with anapparent molecular weight of full-length NME7. FIG. 19F shows a Westernblot in which we used anti-NME7 antibody B9 to probe a gel that wasloaded only with NME1. As can be seen in the figure, antibody B9recognizes NME1 as well as full-length NME7. This is not surprisingbecause like antibody H278, B9 was raised against both A and B domainsof NME7 where the A domain of NME1 is highly homologous to the A domainof NME7_(AB).

FIGS. 20A-20C show graphs of RT-PCR measurements of metastatic markersin cancer cells after being cultured in a serum-free media containingNME7-AB compared to the standard media. FIG. 20A shows SK-OV3, aMUC1-positive ovarian cancer cell line increased expression ofmetastatic markers CXCR4, CDH1 aka E-cadherin, SOX2 and NME7-X1; FIG.20B shows OV-90 a MUC1-negative ovarian cancer cell line increasedexpression of metastatic markers CXCR4 and NME7-X1; FIG. 20C showsMDA-MB a breast cancer cell line that expresses minimal levels of MUC1increased expression of metastatic markers CDH1 aka E-cadherin and SOX2.

FIGS. 21A-21F show photographs of Western blots and description ofcancer cell lines analyzed. For Western blots in FIGS. 21A and 21B, allcancer samples were normalized such that they were loaded onto gel at aconcentration of 40 ug/mL. In FIG. 21A, various cancer cell lines areprobed for the expression of full-length MUC1 using an anti-tandemrepeat monoclonal antibody VU4H5. In FIG. 21B, various cancer cell linesare probed for the expression of cleaved form MUC1* using a polyclonalanti-PSMGFR antibody. FIG. 21C is a description of the cancer cell linesanalyzed. FIG. 21D shows that HER2 positive BT474 breast cancer cells,marked “BT474 (parent cells)” express little to no MUC1 or MUC1* untilthey acquire resistance to Herceptin and other chemotherapy drugs,marked “BTRes1” in figure. Parent cells were made resistant toHerceptin, Taxol, Doxorubicin and cyclophosphamide by culturing thecells in sub-lethal levels of Herceptin. FIG. 21D shows that theexpression level of HER2 has not changed but expression of MUC1* hasdramatically increased as the cells acquired resistance to Herceptin.FIG. 21E shows a graph of the growth of the parent BT474 cells comparedto the drug resistant metastatic cells in response to treatment withHerceptin in the presence or absence of an anti-MUC1* Fab. As can beseen in the figure, the BT474 parent cells show a Herceptinconcentration dependent decrease in cell growth, whereas the twoHerceptin resistant cell lines, BTRes 1 and BTRes2, show no decrease incancer cell growth in response to treatment with Herceptin. However,when treated with an anti-MUC1* Fab, the resistant cell lines show aHerceptin concentration dependent decrease in cancer cell growth. FIG.21F shows a graph of the percent cell death of the parent BT474 cellscompared to the drug resistant BTRes1 cells, in response to treatmentwith Taxol in the presence or absence of an anti-MUC1* Fab.

FIGS. 22A-22E show photographs of Western blots of aco-immunoprecipitation experiment. T47D breast cancer cell extracts wereincubated with an antibody against the MUC1 cytoplasmic tail, Ab-5, or acontrol antibody, IgG, and co-immunoprecipitated. The gels were blottedwith two different commercially available anti-NME7 antibodies B9 (FIG.22A) and CF7 (FIG. 22B). Both gels show unique NME7 bands at ˜33 kDa and˜30 kDa. The gels were stripped and re-probed with an antibody againstthe extracellular domain of MUC1*, anti-PSMGFR (FIG. 22C) and (FIG.22D), which shows that the NME7 species and MUC1* interact. Arecombinant NME7-AB and a recombinant NME7-X1 were mixed together andrun on a gel, then probed with an anti-NME7 antibody, showing that thetwo unique NME7 species that are naturally occurring in breast cancercells and that interact with MUC1* are an NME7-AB-like species andNME7-X1 (FIG. 22E).

FIGS. 23A-23C show photographs of Western blots of aco-immunoprecipitation experiment. Human induced pluripotent stem, iPS7,or embryonic stem, HES3, cell extracts were incubated with an antibodyagainst the MUC1 cytoplasmic tail, Ab-5, or a control antibody, IgG, andco-immunoprecipitated. The gel was blotted with a commercially availableanti-NME7 antibody B9 (FIG. 23A). Both cell types show unique NME7 bandsat ˜33 kDa and ˜30 kDa. The gel was stripped and re-probed with anantibody against the extracellular domain of MUC1*, anti-PSMGFR (FIG.23B), which shows that the NME7 species and MUC1* interact. Arecombinant NME7-AB and a recombinant NME7-X1 were mixed together andrun on a gel, then probed with an anti-NME7 antibody, showing that thetwo unique NME7 species that are naturally occurring in breast cancercells and that interact with MUC1* are an NME7-AB-like species andNME7-X1 (FIG. 23C).

FIG. 24 shows a graph of an ELISA experiment assaying new anti-NME7antibodies for their ability to bind to NME7-AB. NME7-AB is known tobind to the extra cellular domain of MUC1*. The surface of themulti-well plate was coated with a recombinant NME7-AB. Anti-NME7-ABantibodies were separately added to wells. Standard washes wereperformed and visualized by adding an HRP-conjugated secondary antibody.As can be seen, 7 of the 10 new anti-NME7 antibodies bound strongly toNME7-AB.

FIG. 25 shows a graph of an ELISA experiment assaying new anti-NME7antibodies for their ability, or preferably inability, to bind to NME1.The surface of the multi-well plate was coated with a recombinantNME1-S120G dimers, which are also known to bind to the MUC1* extracellular domain. Anti-NME7-AB antibodies were separately added to wells.Standard washes were performed and visualized by adding anHRP-conjugated secondary antibody. As can be seen only one antibodyshowed just minimal binding to NME1.

FIG. 26 shows a graph of an ELISA competitive inhibition assay.NME7-AB/anti-NME7 antibody complexes were made before adding to amulti-well plate coated with MUC1* extra cellular domain peptide,PSMGFR. Recall that NME7-AB has two pseudo-identical domains A and Bthat are each able to bind to MUC1* extra cellular domain. Antibodiesthat bind to the NME7 B3 peptide, which is in the B domain, do not bindto the NME7 A domain. Therefore, only partial inhibition of theNME7-AB/MUC1* interaction is expected.

FIG. 27 shows a graph of an ELISA displacement assay. NME7-AB was firstbound to surface-immobilized MUC1* extra cellular domain peptide on theplate, then disrupted by the addition of anti-NME7 antibodies.

FIG. 28 shows a graph of an ELISA displacement assay. In this case, themulti-well plate was coated with a truncated MUC1* peptide, N-10, whichhas the 10 N-terminal amino acids missing of the PSMGFR sequence.NME7-AB is known to bind to the N-10 peptide. NME7-AB was bound tosurface-immobilized N-10 peptide on the plate, then disrupted by theaddition of anti-NME7 antibodies.

FIG. 29 shows a graph of the amount of RNA present in samples of T47Dbreast cancer cells were cultured in either their normal recommendedmedia, RMPI, serum-free media containing only NME7-AB as the growthfactor at 4 nM, which is optimal, or 8 nM, or serum-free mediacontaining only NME1 S120G dimers as the growth factor at 8 nM; becauseNME1 is a homodimer and NME7-AB is a monomer comprised of twopseudo-identical domains, 8 nM NME1 is the molar equivalent of 4 nMNME7-AB. The cancer cells were cultured in the presence or absence ofanti-NME7 B3 antibodies. In this experiment, floating cells wereseparated from the adherent cells and analyzed separately. Significantdata argues that the floater cells are the cancer stem cells. Anincrease or decrease in the amount of RNA in a sample argues that anagent increased or decreased, respectively, the number of cells in agiven population that were generated.

FIG. 30 shows a graph of a PCR measurement of metastatic marker CXCR4 inT47D breast cancer cells that were cultured in either their normalrecommended media, RPMI, serum-free media containing only NME7-AB as thegrowth factor at 4 nM, which is optimal, or 8 nM, or serum-free mediacontaining only NME1 S120G dimers as the growth factor at 8 nM; becauseNME1 is a homodimer and NME7-AB is a monomer comprised of twopseudo-identical domains, 8 nM NME1 is the molar equivalent of 4 nMNME7-AB. The cancer cells were cultured in the presence or absence ofanti-NME7 B3 antibodies. In this experiment, floating cells wereseparated from the adherent cells and analyzed separately. Significantdata argues that the floater cells are the cancer stem cells. As can beseen in the figure, growth in NME7-AB media increases CXCR4 in thefloater population of cells and anti-NME7 B3 antibodies decreased itsexpression, arguing that anti-NME7 antibodies decreased generation ofcancer stem cells.

FIG. 31 shows a graph of a PCR measurement of stem cell marker andmetastatic marker SOX2 in T47D breast cancer cells that were cultured ineither their normal recommended media, RMPI, serum-free media containingonly NME7-AB as the growth factor at 4 nM, which is optimal, or 8 nM, orserum-free media containing only NME1 S120G dimers as the growth factorat 8 nM; because NME1 is a homodimer and NME7-AB is a monomer comprisedof two pseudo-identical domains, 8 nM NME1 is the molar equivalent of 4nM NME7-AB. The cancer cells were cultured in the presence or absence ofanti-NME7 B3 antibodies. In this experiment, floating cells wereseparated from the adherent cells and analyzed separately. Significantdata argues that the floater cells are the cancer stem cells. As can beseen in the figure, growth in NME7-AB media increases SOX2 expression inthe floater population of cells and anti-NME7 B3 antibodies decreasedits expression, arguing that anti-NME7 antibodies decreased generationof cancer stem cells.

FIG. 32 shows a graph of a PCR measurement of stem cell marker andmetastatic growth factor receptor MUC1 in T47D breast cancer cells thatwere cultured in either their normal recommended media, RMPI, serum-freemedia containing only NME7-AB as the growth factor at 4 nM, which isoptimal, or 8 nM, or serum-free media containing only NME1 S120G dimersas the growth factor at 8 nM; because NME1 is a homodimer and NME7-AB isa monomer comprised of two pseudo-identical domains, 8 nM NME1 is themolar equivalent of 4 nM NME7-AB. The cancer cells were cultured in thepresence or absence of anti-NME7 B3 antibodies. In this experiment,floating cells were separated from the adherent cells and analyzedseparately. Significant data argues that the floater cells are thecancer stem cells. As can be seen in the figure, growth in NME7-AB mediaincreases MUC1 expression in the floater population of cells andanti-NME7 B3 antibodies decreased its expression, arguing that anti-NME7antibodies decreased generation of cancer stem cells.

FIGS. 33A-33B show IVIS photographs of immune compromised nu/nu mice Day6 post tail vein injection of cancer cells. FIG. 33A shows IVISphotographs of mice injected with 500,000 T47D-wt breast cancer cells.FIG. 33B shows IVIS photographs of mice injected with 10,000 T47D breastcancer cells that were grown for 10 days in NME7-AB in a minimal media.The floating cells were collected. These floating cells are referred toherein as cancer stem cells, CSCs. As can be seen in the figure, themice injected with wild type cancer cells show no signs of metastasis.However, the mice injected with 50-times less cells, but cancer stemcells, show that the injected cancer cells are clearly metastasizing.

FIGS. 34A-34D show IVIS photographs of immune compromised nu/nu mice Day10 post tail vein injection of cancer cells. FIG. 34A shows IVISphotographs of mouse injected with 500,000 T47D-wt breast cancer cells.FIG. 34B shows IVIS photographs of mouse injected with 10,000 T47D-CSC(cancer stem cells). FIG. 34C shows IVIS photographs of mouse injectedwith 10,000 T47D-CSC (cancer stem cells) and injected on Day 7 withanti-NME7 antibody. FIG. 34D shows the hand recording of the IVISmeasure of emitted photons. As can be seen in the figure, the mousechosen for treatment is more metastatic than the comparable T47D-CSCmouse. The efficacy of the first antibody injection may have beenblocked by the Day 6 injection of free NME7-AB. Control mouse injectedwith 500,000 T47D-wt cells shows some weak emission of photons that maybe background or surviving cancer cells.

FIG. 35A-35C shows IVIS photographs of immune compromised nu/nu mice Day12 post tail vein injection of cancer cells. FIG. 35A shows IVISphotographs of mouse injected with 500,000 T47D-wt breast cancer cells.FIG. 35B shows that mouse injected with 10,000 T47D-CSC (cancer stemcells) that was not treated with anti-NME7 antibody died from excesstumor burden before IVIS photograph could be taken. FIG. 35C shows IVISphotographs of mouse injected with 10,000 T47D-CSC (cancer stem cells)and injected on Day 7 and Day 10 with anti-NME7 antibody. As can be seenin the figure, the mouse treated with anti-NME7 antibody is clearingaway the cancer metastases. Control mouse injected with 500,000 T47D-wtcells shows less photon emissions indicating fewer surviving cancercells or may be background.

FIGS. 36A-36B shows IVIS photographs of immune compromised nu/nu miceDay 14 post tail vein injection of cancer cells. FIG. 36A shows IVISphotographs of mouse injected with 500,000 T47D-wt breast cancer cells.FIG. 36B shows IVIS photographs of mouse injected with 10,000 T47D-CSC(cancer stem cells) and injected on Day 7, Day 10, and Day 12 withanti-NME7 antibody. As can be seen in the figure, the mouse treated withanti-NME7 antibody nearly completely free of cancer cell metastases.Control mouse injected with 500,000 T47D-wt cells shows no photonemissions.

FIGS. 37A-37V shows time course of IVIS photographs of immunecompromised nu/nu mice from Day 6 to Day 26 post cancer cell tail veininjection. FIGS. 37A, 37C, 37E, 37G, 37I, 37K, 37M and 37O show IVISphotographs of mouse that had been injected Day 0 into the tail veinwith 500,000 T47D-wt cells. FIGS. 37B, 37D, 37F, 37H, 37J, 37L, 37N and37P show IVIS photographs of mouse that had been injected Day 0 into thetail vein with 10,000 T47D cancer stem cells, to which anti-NME7antibody was administered from Day 7 to Day 17, whereupon treatment wassuspended, then resumed on Day 21. FIGS. 37Q, 37R, 37S, 37T, and 37Ushow enlarged IVIS photographs of the treated mouse between Day 17, whenanti-NME7 antibody treatment was suspended, through Day 21, whenantibody treatment was resumed to Day 26. FIG. 37V shows the scale barof the IVIS measurements. As can be seen in this time course, cancercells that had been grown in NME7 readily metastasize and suchmetastasis can be effectively treated, prevented or reversed bytreatment with an antibody that binds to NME7.

FIG. 38A-38C shows time course of IVIS photographs of immune compromisednu/nu mice from Day 6 to Day 19 post injection with either 500,000 T47Dwild type breast cancer cells or 10,000 T47D cancer stem cells. FIG. 38Ashows mice that were injected into the tail vein (i.v.). FIG. 38B showsmice that were injected intra-peritonealy (i.p.). FIG. 38C shows micethat were injected sub-cutaneously (s.c.).

FIGS. 39A-39C shows human lung tissue specimens stained with ananti-NME7 antibody that binds to the B3 peptide. The figure shows lackof NME7 expression on normal tissues, increasing expression of NME7 astumor grade and metastasis increase.

FIGS. 40A-40C shows human small intestine tissue specimens stained withan anti-NME7 antibody that binds to the B3 peptide. The figure showslack of NME7 expression on normal tissues, increasing expression of NME7as tumor grade and metastasis increase.

FIGS. 41A-41D show human colon tissue specimens stained with ananti-NME7 antibody that binds to the B3 peptide. The figure shows lackof NME7 expression on normal tissues, increasing expression of NME7 astumor grade and metastasis increase.

FIGS. 42A-42F shows photographs of female nu/nu mice weighingapproximately 20 g each, which were injected into the tail vein with10,000 Luciferase positive T47D metastatic breast cancer stem cells andtreated with the anti-NME7_(AB) antibody 4A3 also known as 8F9A4A3. Toimage cancer cells, the Luciferase substrate, Luciferin, isintraperitoneally injected 10 minutes before being photographed in IVISinstrument. FIGS. 42A-42C show IVIS photographs with animals face down.FIG. 42D-42F show IVIS photographs with animals face up. FIGS. 42A and42D show control animals injected with phosphate buffered salinesolution. FIGS. 42B and 42E show a prevention model in which animalswere injected with anti-NME7_(AB) antibody 4A3 24 hrs before injectionof the metastatic cancer cells, then approximately every other day for atotal of 12 antibody injections over 22 days. FIGS. 42C and 42F show areversal model in which animals were injected with anti-NME7_(AB)antibody 4A3 24 hrs after injection of the metastatic cancer cells, thenapproximately every other day for a total of 11 antibody injections over20 days.

FIGS. 43A-43F shows photographs of female nu/nu mice weighingapproximately 20 g each, which were injected into the tail vein with10,000 Luciferase positive T47D metastatic breast cancer stem cells andtreated with the anti-NME7_(AB) antibodies 5A1, also known as 8F9A5A1,or 5D4, also known as 5F3A5D4. To image cancer cells, the Luciferasesubstrate, Luciferin, is intraperitoneally injected 10 minutes beforebeing photographed in IVIS instrument. FIGS. 43A-43C show IVISphotographs with animals face down. FIGS. 43D-43F show IVIS photographswith animals face up. FIGS. 43A and 43D show control animals injectedwith phosphate buffered saline solution. FIGS. 43B, 43E, 43C and 43Fshow a prevention model in which animals were injected withanti-NME7_(AB) antibodies 24 hours before injection of the metastaticcancer cells, then approximately every other day for a total of 12antibody injections over 22 days. Images were taken on Day 27.

FIGS. 44A-44D shows photographs of female nu/nu mice that on Day 0 wereinjected into the tail vein with 10,000 Luciferase positive T47Dmetastatic breast cancer stem cells mixed with NME7_(AB) to a finalconcentration of 32 nM. On Day 1 and Day 2 animals were injected intothe tail vein with more 32 nM NME7_(AB), which we have shown increasesmetastases. This is a system to demonstrate reversion of establishedmetastases. On Day 7 animals were treated with individual anti-NME7_(AB)antibodies 8F9A5A1, 8F9A4A3, or 5F3A5D4. FIG. 44A shows control animalsinjected with phosphate buffered saline solution. FIG. 44B shows animalstreated with anti-NME7_(AB) monoclonal antibody 8F9A5A1, also known as5A1. FIG. 44C shows animals treated with anti-NME7_(AB) monoclonalantibody 8F9A4A3, also known as 4A3. FIG. 44D shows animals treated withanti-NME7_(AB) monoclonal antibody 5F3A5D4, also known as 5D4. Greenarrows indicate low antibody dosage (5-7 mg/kg) over the indicatedperiod and Red arrows indicate high dosage (15 mg/kg). As can be seen inthe figure, animals treated with anti-NME7_(AB) antibodies have lessmetastases than the control animals even though many of the animals inthe groups to be treated with antibody have more metastasis before anytreatment. Higher concentrations of anti-NME7_(AB) antibody are moreeffective than low concentrations. For example between Day 11 and Day17, animals were treated with high dose and most of the treated animalshave cleared metastases by about Day 17. However, 1 low dose of antibodyresulted in metastasis recurrence. Animals again respond to high dosetreatment by Day 32.

FIGS. 45A-45B shows photographs of female nu/nu mice that on Day 0 wereinjected sub-cutaneously into the right flank with 10,000 Luciferasepositive T47D metastatic breast cancer stem cells, mixed with NME7_(AB)to a final concentration of 32 nM, then mixed in a 1:1 vol:vol withMatrigel. Tumor engraftment was allowed to progress Day 0-Day 6. Animalswere then treated i.v. by tail vein injection with anti-NME7_(AB)antibodies. Control animals were injected with PBS. FIG. 45A shows IVISphotographs of control animals. FIG. 45B shows IVIS photographs ofanimals injected into tail vein with a cocktail of anti-NME7_(AB)antibodies 5A1, 4A3 and 5D4 to a total concentration of 15 mg/kg.Antibodies or PBS were administered 4 times between Day 7 and Day 18. Ascan be seen in the figure, the anti-NME7_(AB) antibody treated animalsshow less metastases than the control group. In the treated group, 2 ofthe 5 animals have primary tumors that are larger than those in thecontrol group. This could be because the anti-NME7_(AB) antibodiesprevented the spread of the cancer cells, so they remained concentratedin the primary tumor. In this experiment, PCR analysis showed that after11 days in culture with NME7_(AB), the T47D breast cancer cells hadupregulated CXCR4 by 109-fold, OCT4 by 2-fold, NANOG by 3.5-fold andMUC1 by 2.7-fold.

FIGS. 46A-46Q shows photographs of female nu/nu mice that on Day 0 wereinjected sub-cutaneously into the right flank with 10,000 Luciferasepositive T47D metastatic breast cancer stem cells, mixed with NME7_(AB)to a final concentration of 32 nM, then mixed in a 1:1 vol:vol withMatrigel. Tumor engraftment was allowed to progress Day 0-Day 6. Animalswere then treated i.v., by tail vein injection, with anti-NME7_(AB)antibodies. Control animals were injected with PBS. On Day 38 animalswere sacrificed and livers harvested then analyzed by IVIS to detectcancer cells that had metastasized to the liver. FIGS. 46A-46B showwhole body IVIS photographs of control animals that were injected withonly PBS. FIGS. 46C-46D show whole body IVIS photographs of controlanimals that were injected with the anti-NME7_(AB) antibody 5A1. FIGS.46E-46F show whole body IVIS photographs of control animals that wereinjected with the anti-NME7_(AB) antibody 4A3. FIGS. 46G-46H show wholebody IVIS photographs of control animals that were injected with theanti-NME7_(AB) antibody 5D4. FIGS. 46A, 46C, 46E, and 46G are IVISphotographs taken at Day 7 before any treatment. FIGS. 46B, 46D, 46F,and 46H are IVIS photographs taken at Day 31 after anti-NME7_(AB)antibody treatment or mock treatment. As can be seen in the figure,animals in the PBS control group show metastasis (blue dots) in thewhole body IVIS photographs, while animals treated with anti-NME7_(AB)antibodies do not. FIGS. 461-46P show photographs and IVIS photographsof livers and lung harvested from animals after sacrifice. FIGS. 46I,46K, 46M, and 46O are regular photographs. FIGS. 46J, 46L, 46N, and 46Pare IVIS photographs, illuminating the cancer cells that havemetastasized there. As can be seen in the figure, the anti-NME7_(AB)antibodies greatly inhibited metastasis to the liver, which is a primarysite for breast cancer metastasis. FIG. 46Q is a bar graph of themeasured photons emitted and enumerated by IVIS instrument for liversharvested from control animals versus the treated animals.

FIGS. 47A-47F shows photographs of immunofluorescent experiments inwhich various cancer cell lines are stained for the presence ofNME7_(AB). FIG. 47A shows T47D breast cancer cells stained with varyingconcentrations of anti-NME7_(AB) antibody 5D4. FIG. 47B shows ZR-75-1breast cancer cells, also known as 1500s, stained with varyingconcentrations of anti-NME7_(AB) antibody 5D4. FIG. 47C shows H1975non-small cell lung cancer cells stained with varying concentrations ofanti-NME7_(AB) antibody 5D4. FIG. 47D shows H292 non-small cell lungcancer cells stained with varying concentrations of anti-NME7_(AB)antibody 5D4. FIG. 47E shows HPAFII pancreatic cancer cells stained withvarying concentrations of anti-NME7_(AB) antibody 5D4. FIG. 47F showsDU145 prostate cancer cells stained with varying concentrations ofanti-NME7_(AB) antibody 5D4. As can be seen in the figure, all thecancer cell lines we tested show strong and membranous staining forNME7_(AB). The monoclonal antibody used in these experiments was 5D4. Inparallel, NME7_(AB) antibodies 5A1 and 4A3 were used to stain the samecell lines and produced the same results.

FIGS. 48A-48I shows photographs of immunofluorescent experiments inwhich various lung cancer cell lines are stained for the presence ofNME7_(AB). FIGS. 48A-48C shows H1975 non-small cell lung cancer cells,which are an adenocarcinoma, stained with varying concentrations ofanti-NME7_(AB) antibody 5D4. FIG. 48A is an overlay of DAPI andanti-NME7_(AB) staining. FIG. 48B shows anti-NME7_(AB) staining alone.FIG. 48C is a magnified view of the overlay of DAPI and anti-NME7_(AB)staining. FIGS. 48D-48F shows H292 non-small cell lung cancer cells,which are a mucoepidermoid pulmonary carcinoma, stained with varyingconcentrations of anti-NME7_(AB) antibody 5D4. FIG. 48D is an overlay ofDAPI and anti-NME7_(AB) staining. FIG. 48E shows anti-NME7_(AB) stainingalone. FIG. 48F is a magnified view of the overlay of DAPI andanti-NME7_(AB) staining. FIGS. 48G-48I shows H358 non-small cell lungcancer cells, which are a metastatic bronchioalveolar carcinoma, stainedwith varying concentrations of anti-NME7_(AB) antibody 5D4. FIG. 48G isan overlay of DAPI and anti-NME7_(AB) staining. FIG. 48H showsanti-NME7_(AB) staining alone. FIG. 48I is a magnified view of theoverlay of DAPI and anti-NME7_(AB) staining.

FIG. 49A-49I shows PCR graphs of cancer cell lines, breast T47D, LungH1975, lung H358 and pancreatic HPAFII before and after culture inNME7_(AB). FIG. 49A measured breast metastatic marker CXCR4. FIG. 49Bmeasured stem cell marker OCT4. FIG. 49C measured metastatic markerALDH1. FIG. 49D measured stem cell marker SOX2. FIG. 49E measured stemcell marker NANOG. FIG. 49F measured marker CDH1, also known asE-cadherin. FIG. 49G measured metastatic marker CD133. FIG. 49H measuredstem cell marker ZEB2. FIG. 49I measured stem, cancer and metastaticmarker MUC1. The floater cells, also known as tumor spheres become ableto grow anchorage independently and show markers of metastasis that aremore elevated than the adherent cells. Animals injected with cancer stemcells are those injected with the NME7_(AB) grown floater cells. As canbe seen in the figure markers of metastasis, stem cell markers, ormarkers of epithelial to mesenchymal transition (EMT) are elevated afterculture in NME7_(AB), indicating a transition to a more metastaticstate.

FIG. 50A-50D shows IVIS photographs of NSG mice injected into the tailvein with 10,000 cancer cells that were either NCI-H358 parent cells orNCI-H358 cells after 10 days in culture with NME7_(AB). FIGS. 50A and50C show IVIS photographs of the mouse that was injected with theNCI-H358 lung cancer cells that had been grown in NME7_(AB) for 10 days.FIGS. 50B and 50D show IVIS photographs of the mouse that was injectedwith the parental NCI-H358 cells. FIGS. 50A and 50B show the IVISphotographs where mice are imaged face down. FIGS. 50C and 50D show theIVIS photographs where mice are imaged face up. As can be seen in thefigure, the NME7_(AB) grown cells have greatly increased metastaticpotential.

FIG. 51 shows PCR graph of a MUC1 negative prostate cancer line PC3before and after 2 or 3 passages in culture in either dimeric NM23-H1,also known as NME1, or NME7_(AB). The graph shows the fold difference inmarkers of stem cells, cancer cells as well as metastatic markers. Ascan be seen in the figure, repeated culture in NME1 or NME7_(AB) inducesupregulation of stem, cancer and metastatic markers but also upregulatesexpression of MUC1 by 5-8 times.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

In the present application, “a” and “an” are used to refer to bothsingle and a plurality of objects.

As used herein, “about” or “substantially” generally provides a leewayfrom being limited to an exact number. For example, as used in thecontext of the length of a polypeptide sequence, “about” or“substantially” indicates that the polypeptide is not to be limited tothe recited number of amino acids. A few amino acids add to orsubtracted from the N-terminus or C-terminus may be included so long asthe functional activity such as its binding activity is present.

As used herein, administration “in combination with” one or more furthertherapeutic agents includes simultaneous (concurrent) and consecutiveadministration in any order.

As used herein, “amino acid” and “amino acids” refer to all naturallyoccurring L-α-amino acids. This definition is meant to includenorleucine, ornithine, and homocysteine.

As used herein, in general, the term “amino acid sequence variant”refers to molecules with some differences in their amino acid sequencesas compared to a reference (e.g. native sequence) polypeptide. The aminoacid alterations may be substitutions, insertions, deletions or anydesired combinations of such changes in a native amino acid sequence.

Substitutional variants are those that have at least one amino acidresidue in a native sequence removed and a different amino acid insertedin its place at the same position. The substitutions may be single,where only one amino acid in the molecule has been substituted, or theymay be multiple, where two or more amino acids have been substituted inthe same molecule.

Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Also included within the scope of the invention areproteins or fragments or derivatives thereof which exhibit the same orsimilar biological activity and derivatives which are differentiallymodified during or after translation, e.g., by glycosylation,proteolytic cleavage, linkage to an antibody molecule or other cellularligand, and so on.

Insertional variants are those with one or more amino acids insertedimmediately adjacent to an amino acid at a particular position in anative amino acid sequence. Immediately adjacent to an amino acid meansconnected to either the α-carboxy or α-amino functional group of theamino acid.

Deletional variants are those with one or more amino acids in the nativeamino acid sequence removed. Ordinarily, deletional variants will haveone or two amino acids deleted in a particular region of the molecule.

As used herein, “fragments” or “functional derivatives” refers tobiologically active amino acid sequence variants and fragments of thepolypeptide of the present invention, as well as covalent modifications,including derivatives obtained by reaction with organic derivatizingagents, post-translational modifications, derivatives withnonproteinaceous polymers, and immunoadhesins.

As used herein, “carriers” include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe pharmaceutically acceptable carrier is an aqueous pH bufferedsolution. Examples of pharmaceutically acceptable carriers includewithout limitation buffers such as phosphate, citrate, and other organicacids; antioxidants including ascorbic acid; low molecular weight (lessthan about 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

As used herein “pharmaceutically acceptable carrier and/or diluent”includes any and all solvents, dispersion media, coatings antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, use thereofin the therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active material for the treatment ofdisease in living subjects having a diseased condition in which bodilyhealth is impaired.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form. A unit dosageform can, for example, contain the principal active compound in amountsranging from 0.5 μg to about 2000 mg. Expressed in proportions, theactive compound is generally present in from about 0.5 μg/ml of carrier.In the case of compositions containing supplementary active ingredients,the dosages are determined by reference to the usual dose and manner ofadministration of the said ingredients.

As used herein, “vector”, “polynucleotide vector”, “construct” and“polynucleotide construct” are used interchangeably herein. Apolynucleotide vector of this invention may be in any of several forms,including, but not limited to, RNA, DNA, RNA encapsulated in aretroviral coat, DNA encapsulated in an adenovirus coat, DNA packaged inanother viral or viral-like form (such as herpes simplex, andadeno-structures, such as polyamides.

As used herein, “host cell” includes an individual cell or cell culturewhich can be or has been a recipient of a vector of this invention. Hostcells include progeny of a single host cell, and the progeny may notnecessarily be completely identical (in morphology or in total DNAcomplement) to the original parent cell due to natural, accidental, ordeliberate mutation and/or change.

As used herein, “subject” is a vertebrate, preferably a mammal, morepreferably a human.

As used herein, “mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep,pigs, and so on. Preferably, the mammal is human.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. “Treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. “Palliating” a disease means that theextent and/or undesirable clinical manifestations of a disease state arelessened and/or the time course of the progression is slowed orlengthened, as compared to a situation without treatment.

As used herein, “A1” peptide, “A2” peptide, “B1” peptide, “B2” peptideand “B3” peptide refer to peptides derived from NME7 that are used togenerate or select antibodies that bind to human NME7_(AB), but not (orsignificantly less) to human NME1. The peptides used to generate theseantibodies are common to both NME7_(AB) and NME7-X1, and are set forthas below.

A1 is NME7A peptide 1 (A domain): (SEQ ID NO: 141) MLSRKEALDFHVDHQSA2 is NME7A peptide 2 (A domain): (SEQ ID NO: 142) SGVARTDASESB1 is NME7B peptide 1 (B domain): (SEQ ID NO: 143) DAGFEISAMQMFNMDRVNVEB2 is NME7B peptide 2 (B domain): (SEQ ID NO: 144) EVYKGVVTEYHDMVTEB3 is NME7B peptide 3 (B domain): (SEQ ID NO: 145)AIFGKTKIQNAVHCTDLPEDGLLEVQYFF

Further, for the sake of clarity, NME7A (with capital letter “A”) refersto the subunit A portion of NME7. NME7a (with small letter “a”) refersto the full-length NME7 that is described elsewhere in this application.And, NME7B (with capital letter “B”) refers to the subunit B portion ofNME7. NME7b (with small letter “b”) refers to a species of NME7 that ispartially devoid of the DM10 region, which is described elsewhere inthis application.

As used herein, the term “antibody-like” means a molecule that may beengineered such that it contains portions of antibodies but is not anantibody that would naturally occur in nature. Examples include but arenot limited to CAR (chimeric antigen receptor) T cell technology and theYlanthia® technology. The CAR technology uses an antibody epitope fusedto a portion of a T cell so that the body's immune system is directed toattack a specific target protein or cell. The Ylanthia® technologyconsists of an “antibody-like” library that is a collection of synthetichuman fabs that are then screened for binding to peptide epitopes fromtarget proteins. The selected Fab regions can then be engineered into ascaffold or framework so that they resemble antibodies.

As used herein, an “effective amount of an agent to inhibit an NMEfamily member protein” refers to the effective amount of the agent inhindering the activating interaction between the NME family memberprotein and its cognate receptor such as

As used herein, “NME derived fragment” refers to a peptide sequence thatis either a fragment of the NME or is highly homologous to the peptidesequence that is a fragment of the NME.

As used herein, the “MUC1*” extra cellular domain is defined primarilyby the PSMGFR sequence (GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA(SEQ ID NO:6)). Because the exact site of MUC1 cleavage depends on theenzyme that clips it, and that the cleavage enzyme varies depending oncell type, tissue type or the time in the evolution of the cell, theexact sequence of the MUC1* extra cellular domain may vary at theN-terminus.

As used herein, the term “PSMGFR” is an acronym for Primary Sequence ofMUC1 Growth Factor Receptor as set forth asGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:6). In thisregard, the “N-number” as in “N-10 PSMGFR” or simply “N-10”, “N-15PSMGFR” or simply “N-15”, or “N-20 PSMGFR” or simply “N-20” refers tothe number of amino acid residues that have been deleted at theN-terminal end of PSMGFR. Likewise “C-number” as in “C-10 PSMGFR” orsimply “C-10”, “C-15 PSMGFR” or simply “C-15”, or “C-20 PSMGFR” orsimply “C-20” refers to the number of amino acid residues that have beendeleted at the C-terminal end of PSMGFR. A mixture of deletions andadditions is also possible. For instance, N+20/C-27 refers to a peptidefragment of wild-type MUC1 in which 20 amino acids are added to thePSMGFR at the N-terminus and 27 amino acids are deleted from theC-terminus.

As used herein, the “extracellular domain of MUC1*” refers to theextracellular portion of a MUC1 protein that is devoid of the tandemrepeat domain. In most cases, MUC1* is a cleavage product wherein theMUC1* portion consists of a short extracellular domain devoid of tandemrepeats, a transmembrane domain and a cytoplasmic tail. The preciselocation of cleavage of MUC1 is not known perhaps because it appearsthat it can be cleaved by more than one enzyme. The extracellular domainof MUC1* will include most of the PSMGFR sequence but may have anadditional 10-20 N-terminal amino acids.

As used herein, “high homology” is considered to be at least 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97%identity in a designated overlapping region between any twopolypeptides.

As used herein, “NME family proteins” or “NME family member proteins”,numbered 1-10, are proteins grouped together because they all have atleast one NDPK (nucleotide diphosphate kinase) domain. In some cases,the NDPK domain is not functional in terms of being able to catalyze theconversion of ATP to ADP. NME proteins were formerly known as NM23proteins, numbered H1 and H2. Recently, as many as ten (10) NME familymembers have been identified. Herein, the terms NM23 and NME areinterchangeable. Herein, terms NME1, NME2, NME5, NME6, NME7, NME8 andNME9 are used to refer to the native protein as well as NME variants. Insome cases these variants are more soluble, express better in E. coli orare more soluble than the native sequence protein. For example, NME7 asused in the specification can mean the native protein or a variant, suchas NME7_(AB) that has superior commercial applicability becausevariations allow high yield expression of the soluble, properly foldedprotein in E. coli. NME7_(AB) consists primarily of the NME7 A and Bdomains but is devoid of most of the DM10 domain (SEQ ID NO:39), whichis at the N-terminus of the native protein. “NME1” as referred to hereinis interchangeable with “NM23-H1”. It is also intended that theinvention not be limited by the exact sequence of the NME proteins. Themutant NME1-5120G, also called NM23-5120G, are used interchangeablythroughout the application. The 5120G mutants and the P96S mutant arepreferred because of their preference for dimer formation, but may bereferred to herein as NM23 dimers, NME1 dimers, or dimeric NME1, ordimeric NM23.

NME7 as referred to herein is intended to mean native NME7 having amolecular weight of about 42 kDa.

A “family of NME7” refers to full length NME7 as well as naturallyoccurring or artificially created cleaved form having a molecular weightabout 30 kDa, 33 kDa, or a cleaved form having a molecular weight ofabout 25 kDa, a variant devoid or partially devoid of the DM10 leadersequence (SEQ ID NO:162), which is NME7 amino acids 1-91 of NME7represented by SEQ ID NO:82 or 147, such as NME7b, NME7-X1, NME7_(AB) ora recombinant NME7 protein, or variants thereof whose sequence may bealtered to allow for efficient expression or that increase yield,solubility or other characteristics that make the NME7 more effective orcommercially more viable. The “family of NME7” may also include“NME7_(AB)-like” protein, which is a protein in the range of 30 to 33kDa that is expressed in cancer cells.

As used herein, an “an agent that maintains stem cells in the naïvestate or reverts primed stem cells to the naïve state” refers to aprotein, small molecule or nucleic acid that alone or in combinationmaintains stem cells in the naïve state, resembling cells of the innercell mass of an embryo. Examples include but are not limited to humanNME1 dimers, bacterial, fungal, yeast, viral or parasitic NME proteinsthat have high sequence identity to human NME proteins, especially NME1,NME7, NME7-X1, NME7_(AB), NME6, 2i (Silva J et al, 2008; Hanna et al,2010), 5i (Theunissen T W et al, 2014), nucleic acids such as siRNA thatsuppress expression of MBD3, CHD4 (Rais Y1 et al, 2013), BRD4, or JMJD6(Liu W et al 2013).

As used herein, the terms “NME7_(AB)”, “NME7_(AB)” and “NME-AB” are usedinterchangeably.

As used herein, an “an agent that promotes pluripotency” or “revertssomatic cells to a stem-like or cancer-like state” refers to a protein,small molecule or nucleic acid that alone or in combination inducesexpression of or suppresses expression of certain genes such that thegenetic signature shifts to one that more closely resembles stem cellsor cancer cells. Examples include but are not limited to NME1 dimers,NME7, NME7-X1, NME7_(AB), 2i, 5i, nucleic acids such as siRNA thatsuppress expression of MBD3, CHD4, BRD4, or JMJD6, microbial NMEproteins that have high sequence homology to human NME1, NME2, NME5,NME6, NME7, NME8, or NME9, preferably with the regions that house NDPKdomains.

As used herein, in reference to an agent being referred to as a “smallmolecule”, it may be a synthetic chemical or chemically based moleculehaving a molecular weight between 50 Da and 2000 Da, more preferablybetween 150 Da and 1000 Da, still more preferably between 200 Da and 750Da.

As used herein, in reference to an agent being referred to as a “naturalproduct”, it may be chemical molecule or a biological molecule, so longas the molecule exists in nature.

As used herein, FGF, FGF-2 or bFGF refer to fibroblast growth factor (XuR H et al, 2005; Xu C et al, 2005).

As used herein, “Rho associated kinase inhibitors” may be smallmolecules, peptides or proteins (Rath N, et al, 2012). Rho kinaseinhibitors are abbreviated here and elsewhere as ROCi or ROCKi, or Ri.The use of specific rho kinase inhibitors are meant to be exemplary andcan be substituted for any other rho kinase inhibitor.

As used herein, the term “cancer stem cells” or “tumor initiating cells”refers to cancer cells that express levels of genes that have beenlinked to a more metastatic state or more aggressive cancers. The terms“cancer stem cells” or “tumor initiating cells” can also refer to cancercells for which far fewer cells are required to give rise to a tumorwhen transplanted into an animal. Cancer stem cells and tumor initiatingcells are often resistant to chemotherapy drugs.

As used herein, the terms “stem/cancer”, “cancer-like”, “stem-like”refers to a state in which cells acquire characteristics of stem cellsor cancer cells, share important elements of the gene expression profileof stem cells, cancer cells or cancer stem cells. Stem-like cells may besomatic cells undergoing induction to a less mature state, such asincreasing expression of pluripotency genes. Stem-like cells also refersto cells that have undergone some de-differentiation or are in ameta-stable state from which they can alter their terminaldifferentiation. Cancer like cells may be cancer cells that have not yetbeen fully characterized but display morphology and characteristics ofcancer cells, such as being able to grow anchorage-independently orbeing able to give rise to a tumor in an animal.

As used herein, “spacers” or “linkers” of different lengths can beincorporated anywhere in the peptide. Spacer attachment is usuallythrough an amide linkage but other functionalities are possible.

NME, NME7 and Protein Family of NME7

The present inventors discovered that NME7 and NME7-X1 are highlyexpressed in early human stem cells and also in most cancer cells (FIG.17 , FIG. 18 , FIG. 19A-FIG. 19F, FIG. 22 , FIG. 23 , FIG. 39 , FIG. 40, FIG. 41 , FIG. 47 , FIG. 48 . FIG. 17 shows a graph of RT-PCRmeasurement of the expression of NME7-X1 in a panel of human stem cellsand cancer cells. FIG. 18 shows a graph of RT-PCR measurement of theexpression of NME7, NME7a, NME7b and NME7-X1 in a panel of human stemcells and cancer cells. NME7a is full-length NME7, NME7b is missing asmall portion of the DM10 domain, NME7-X1 is missing all of the DM10domain and a small portion of the N-terminus of the first NDPK A domain.The bar labeled NME7 means that primers were used that detected bothNME7a and NME7b. FIGS. 19A-19F show photographs of Western blots inwhich various cancer cell lines are probed for expression of NME7species using antibodies generated by immunization with NME7 derivedshort peptides. FIG. 19A shows Western probed with antibody of theinvention #52 which binds to NME7 derived peptide A1. FIG. 19B showsWestern probed with antibody of the invention #56 which binds to NME7derived peptide B1. FIG. 19C shows Western probed with antibody of theinvention #61 which binds to NME7 derived peptide B3. FIGS. 22A-22E showphotographs of Western blots of a co-immunoprecipitation experiment.T47D breast cancer cell extracts were incubated with an antibody againstthe MUC1 cytoplasmic tail, Ab-5, or a control antibody, IgG, andco-immunoprecipitated. The gels were blotted with two differentcommercially available anti-NME7 antibodies B9 (FIG. 22A) and CF7 (FIG.22B). Both gels show unique NME7 bands at ˜33 kDa and ˜30 kDa. The gelswere stripped and re-probed with an antibody against the extracellulardomain of MUC1*, anti-PSMGFR (FIG. 22C) and (FIG. 22D), which shows thatthe NME7 species and MUC1* interact. A recombinant NME7-AB and arecombinant NME7-X1 were mixed together and run on a gel, then probedwith an anti-NME7 antibody, showing that the two unique NME7 speciesthat are naturally occurring in breast cancer cells and that interactwith MUC1* are an NME7-AB-like species and NME7-X1 (FIG. 22E). FIGS.23A-23C show photographs of Western blots of a co-immunoprecipitationexperiment. Human induced pluripotent stem, iPS7, or embryonic stem,HES3, cell extracts were incubated with an antibody against the MUC1cytoplasmic tail, Ab-5, or a control antibody, IgG, andco-immunoprecipitated. The gel was blotted with a commercially availableanti-NME7 antibody B9 (FIG. 23A). Both cell types show unique NME7 bandsat ˜33 kDa and ˜30 kDa. The gel was stripped and re-probed with anantibody against the extracellular domain of MUC1*, anti-PSMGFR (FIG.23B), which shows that the NME7 species and MUC1* interact. Arecombinant NME7-AB and a recombinant NME7-X1 were mixed together andrun on a gel, then probed with an anti-NME7 antibody, showing that thetwo unique NME7 species that are naturally occurring in breast cancercells and that interact with MUC1* are an NME7-AB-like species andNME7-X1 (FIG. 23C). FIGS. 39A-39C shows human lung tissue specimensstained with an anti-NME7 antibody that binds to the B3 peptide. Thefigure shows lack of NME7 expression on normal tissues, increasingexpression of NME7 as tumor grade and metastasis increase. FIGS. 40A-40Cshows human small intestine tissue specimens stained with an anti-NME7antibody that binds to the B3 peptide. The figure shows lack of NME7expression on normal tissues, increasing expression of NME7 as tumorgrade and metastasis increase. FIGS. 41A-41D show human colon tissuespecimens stained with an anti-NME7 antibody that binds to the B3peptide. The figure shows lack of NME7 expression on normal tissues,increasing expression of NME7 as tumor grade and metastasis increase.FIG. 47 and FIG. 48 show immunofluorescent photographs showing that NME7is secreted by and binds to an extra cellular receptor of a wide varietyof cancer cell lines.

Further, we demonstrated that like NM23-H1, NME7 binds to and dimerizesthe MUC1* growth factor receptor on both stem cells and cancer cells(FIG. 1 ). FIG. 5 shows a sequence alignment of NME1 and NME7 A and Bdomains.

The inventors recently discovered that NME7 is a primitive form of NME1(NM23-H1) that is expressed in very early embryonic stem cells. NME7 iseither not expressed at all, or is expressed at extremely low levels, inadult tissues. However, the inventors discovered that NME7 is expressedat high levels in cancerous cells and tissues and at even higher levelsin metastatic cancer cells and tissues. A cleaved form of NME7 may be asecreted form allowing it to bind to and activate extracellularreceptors. We detect full-length NME7, MW 42 kDa, as well as NME7species that are approximately 33 kDa and 30 kDa. The 33 kDa and 30 kDaspecies are secreted from cancer cells. Western blots detect full-lengthNME7 in cell lysates, but smaller 30-33kDaNME7 species in theirconditioned media. Western blots probed with either an antibody thatrecognizes NME7 or an antibody that only recognizes the DM10 domain showthat the lower molecular weight NME7 species that are secreted into theconditioned media are devoid of the DM10 domain. These data areconsistent with the idea that naturally occurring NME7 species arecomparable to the recombinant NME7_(AB) we generated as they have nearlythe same molecular weight, both are secreted and are both devoid of the91 amino acids of the DM10 domain which may keep the protein retainedwithin the cell.

We discovered a new NME7 isoform, NME7-X1, and also discovered that itis over-expressed in stem cells and cancer cells and is particularlyover-expressed in prostate cancers (FIG. 17 , FIG. 18 , FIG. 19 , andFIG. 22 ). NME7-X1, molecular weight ˜30 kDa, comprises NME7 amino acids125-376, whereas the recombinant NME7_(AB), molecular weight ˜33 kDa,that we generated spans amino acids 92-376, so includes 33 moreN-terminal amino acids. NME7b spans amino acids 37-376 and is devoid ofonly 37 amino acids of the DM10 domain is also overexpressed in prostatecancers (FIG. 18 ). We generated a human recombinant NME7-X1 and showthat it is the secreted 30 kDa NME7 species in cancer cells that runsjust lower than a naturally occurring ˜33kDaNME7 species that appears tobe a naturally occurring “NME7_(AB)-like” protein that is a cleavageproduct or alternative isoform.

We tested a panel of cancer cell lines and found that they express highlevels of NME7 and lower molecular weight species that may betruncations similar to NME7_(AB), such as NME7_(AB)-like protein, oralternate isoforms such as NME7-X1.

Whereas NM23-H1 (aka NME1) has to be a dimer, NME7 is a monomer with twobinding sites for MUC1* extracellular domain. We generated a recombinanthuman NME7 that is devoid of the DM10 domain, which we call NME7_(AB). Asandwich ELISA binding assay that shows that a recombinant NME7_(AB)simultaneously binds to two PSMGFR peptides wherein the extracellulardomain of MUC1* is comprised of most or all of the PSMGFR sequence (FIG.1 ). In a nanoparticle binding assay, NME7 was also shown to be able tobind to and dimerize the PSMGFR portion of the MUC1* extracellulardomain.

Agents that disable NME7, block its interaction with its bindingpartners or suppress its expression are potent anti-cancer therapeutics.Such agents may be antibodies, small molecules or nucleic acids. Theymay act on NME7 directly, on molecules that regulate NME7 expression, oron enzymes that cleave NME7 to cancer-promoting forms.

We discovered that like NM23-H1 dimers, a recombinant NME7_(AB) monomerwas fully able to support pluripotent human stem cell growth in theabsence of any other growth factor, cytokine or serum. Competitivelyinhibiting the interaction between NME7 and MUC1* extracellular domain,comprised essentially of the PSMGFR sequence, induced differentiation ofstem cells, showing that it is the interaction of NME7 and MUC1* thatpromotes stem cell growth and inhibits differentiation.

Next, we showed that NME7_(AB) alone is also able to fully support humancancer cell growth. NME7_(AB), when added to regular cancer cell growthmedia, stimulated cancer cell growth and in particular the growth ofMUC1-positive and MUC1*-positive cancer cells. Inhibiting theinteraction of NME7 with MUC1* inhibited cancer cell growth. Blockingthe MUC1* growth factor receptor with an anti-MUC1* Fab potentlyinhibited cancer cell growth. Similarly, antibodies that bind to NME7inhibit cancer cell growth. In one example of inhibition of cancergrowth by anti-NME7 antibody, the polyclonal antibody was generated fromimmunizing an animal with the portion of NME7 that spans amino acids100-376 (FIG. 12 and FIG. 13 ). However, we found that antibodiesgenerated from immunizing with shorter peptides from NME7_(AB) or fromNME7-X1 also inhibit cancer growth. In particular, they inhibit thegrowth of MUC1 and MUC1*-positive cancers. Anti-NME7 antibodies of theinvention inhibited the formation of the non-adherent “floater” cellsthat are able to form tumor spheres and which can travel from primarytumor and metastasize (FIG. 14 , FIG. 16 , FIG. 29 ). Anti-NME7antibodies of the invention inhibited the upregulation of metastatic andstem cell markers, now believed to also be characteristic of metastasis(FIG. 15 , FIG. 30 , FIG. 31 , FIG. 32 ).

NME7 Causes Cancer Metastasis

The inventors further discovered that culturing cancer cells in aminimal media containing NME7_(AB) induced a wide variety of cancercells to become transformed to a more metastatic state. Evidence of thisinduced metastatic state include a change from adherent cell growth tonon-adherent cell growth, aka, “floater” cells and accompanyingup-regulation of specific metastatic markers that were especiallyupregulated in the floating cells. These metastatic markers that areupregulated after culture in NME7_(AB) include but are not limited toCXCR4, CHD1 aka E-cadherin, MUC1, ALDH1, CD44, and pluripotent stem cellmarkers such as OCT4, SOX2, NANOG, KLF2/4, FOXa2, TBX3, ZEB2 and c-Myc(FIG. 2 , FIG. 3 , FIG. 20 , FIG. 49 , FIG. 51 ). Cancer cells culturedin NME7_(AB) had dramatically higher engraftment rates when xenograftedinto test animals, which were over 90%. In addition, very low numbers ofimplanted cancer cells formed tumors in the test animals, which isevidence that NME7_(AB) had transformed them into cancer stem cells alsoknown as metastatic cancer cells. Cancer cells cultured in NME7_(AB) andinjected into the tail vein of NOD/SCID/GAMMA mice bearing estrogenrelease pellets metastasized in animals from low numbers of cellscompared to the parent cells, grown in regular media (FIG. 33 -FIG. 38). Because cancer cells make either an NME7 cleavage product oralternative isoform that is essentially equivalent to NME7_(AB), themethods described here are not limited to using NME7_(AB); other NME7species could work as well. For example, we discovered another NME7isoform, NME7-X1, is expressed by cancer cells. It is identical to ourrecombinant NME7_(AB) with the exception that the X1 isoform is missing33 amino acids from the N-terminus. NME7-X1 is expected to function likeNME7_(AB). “NME7_(AB)-like” protein has also been detected in cancercells as being about 33 Da species.

We note that the inventors' previous work showed that NME7_(AB) alone isable to revert human stem cells to an earlier naïve state. We discoveredthat culturing cancer cells in the presence of other reagents that makestem cells revert to a more naïve state, makes the cancer cellstransform to a more metastatic state. We demonstrated that culturingcancer cells NME7_(AB) (FIG. 2 ), or in dimeric NME1 (FIG. 3 ), or “2i”inhibitors (FIG. 4 ), are each able to transform regular cancer cellsinto metastatic cancer cells, which are also called cancer stem cells“CSCs” or tumor initiating cells “TICs”. However, NME7_(AB) inducedcancer cells to enter a more metastatic state better than NME1, alsoknown as NM23-H1, which was better than 2i.

2i is the name given to two biochemical inhibitors that researchersfound made human stem cells revert to a more naïve state. 2i are MEK andGSK3-beta inhibitors PD0325901 and CHIR99021, which are added to culturemedium to final concentrations of about 1 mM and 3 mM, respectively.NME7_(AB) and NME7-X1 are at a final concentration of about 4 nM whenadded to separate batches of minimal medium to make cancer cellstransform to metastatic cells, although lower and higher concentrationsalso work well in the range of about 1 nM to 16 nM. Human or bacterialNME1 dimers are used at a final concentration of 4 nM to 32 nM, with 16nM typically used in these experiments, wherein the human NME bears theS120G mutation. Lower concentrations may be required if using wild type.It is not intended that these exact concentrations are important. It isimportant that the NME1 proteins are dimers and the range ofconcentrations over which this happens is in the low nanomolar rangealthough certain mutations allow higher concentrations to remain asdimers. Similarly, the concentrations of NME7 proteins can vary.NME7_(AB) and NME7-X1 are monomers and concentrations used to transformcancer cells to metastatic cells should allow the proteins to remain asmonomers.

In addition to NME7, NME7_(AB), NME7-X1, and the 2i inhibitors MEKi andGSK3i, other reagents and inhibitors have been shown by others to causestem cells to revert to a more naïve state. These inhibitors, “i's”include JNKi, p38i, PKCi, ROCKi, BMPi, BRAFi, SRCi as well as growthfactors activing and LIF (Gafni et al 2013, Chan et al 2013, Valamehr etal 2014, Ware et al 2014, Theunissen et al 2014). These reagents canalso be used to make cancer cells progress to a more metastatic state.Cells that have been induced to transform to a more metastatic stateusing any single factor or combination of the inhibitors or growthfactors, that make stem cells revert to a more naïve state, can then beused as discovery tools to identify or test drugs to treat or preventcancer metastasis.

Various molecular markers have been proposed as being indicators ofmetastatic cancer cells. Different cancer types may have differentmolecules that are up-regulated. For example, the receptor CXCR4 isup-regulated in metastatic breast cancers while E-cadherin, also knownas CHD1, is up-regulated more in metastatic prostate cancers. Inaddition to these specific metastasis markers, typical markers ofpluripotency such as OCT4, SOX2, NANOG, and KLF4 are up-regulated ascancers become metastatic. The starting cancer cells and the latermetastatic cancer cells are assayed by PCR to measure expression levelsof these genes. We demonstrated that these cancer cells, cultured inagents such as NME7_(AB) that cause them to be transformed to a moremetastatic state, as evidenced by increased expression of metastaticmarkers and pluripotent stem cell markers, function as metastatic cancercells.

A functional test of whether or not a population of cancer cells ismetastatic is to implant very low numbers, e.g. 200, of the cells inimmuno-compromised mice and see if they develop into a tumor. Typically5-6 million cancer cells are required to form a tumor in animmuno-compromised mouse. We showed that as few as 50 of the NME-inducedmetastatic cancer cells formed tumors in mice. In addition, mice thatwere injected throughout the test period with human NME7_(AB), NME1, orNME7-X1 developed remote metastases.

In one particular experiment, T47D human breast cancer cells werecultured in standard RPMI media for 14 days with media changes every 48hours and passed by trypsinization when approximately 75% confluent. Thecells were then plated into 6-well plates and cultured in minimal stemcell media (see Example 1) that was supplemented with 4 nM NME7_(AB) B.Media was changed every 48 hours. By about Day 4, some cells becomedetached from the surface and float. Media is carefully changed so as toretain the “floaters” as these are the cells that have the highestmetastatic potential as evidence by RT-PCR measurement of metastaticmarkers. On Day 7 or 8, the floaters are harvested and counted. Samplesare retained for RT-PCR measurement. The key marker measured is CXCR4,which is up-regulated by 40-200-times after being briefly cultured inNME7_(AB).

The freshly harvested floater metastatic cells were xenografted into theflank of female nu/nu athymic mice that have been implanted with 90-dayslow release estrogen pellets. Floater cells were xenografted with10,000, 1,000, 100 or 50 cells each. Half of the mice in each group of 6were also injected daily with 32 nM NME7_(AB) near the originalimplantation site. The parent T47D cells that were cultured in RPMImedia without NME7_(AB) were also implanted into mice at 6 million,10,000 or 100 as controls. Mice implanted with the NME7-induced floatercells developed tumors even when as few as 50 cells were implanted. Micethat were implanted with the floater cells and that received dailyinjections of NME7_(AB) also developed remote tumors or remotemetastases in various organs. 11 out of the 12 mice, or 92%, that wereinjected with human NME7_(AB) after implantation of the NME7_(AB)cultured cancer cells developed tumors at the injection site. Only 7 outof the 12 mice, or 58%, that were not injected with human NME7_(AB)after implantation developed tumors. 9 out of the 11 mice, or 82%, thatexhibited tumors and were injected with human NME7_(AB) developedmultiple tumors remote from the injection site. None of the mice thatwere not injected with NME7_(AB) developed multiple, visible tumors.

After sacrifice, RT-PCR and Western blots showed that the remote bumpson the mice injected with NME7_(AB) were indeed human breast tumors.Similar analysis of their organs showed that in addition to remotebumps, mice had randomly metastasized to the liver and lung with humanbreast cancer characteristic of the human breast cancer cells that wereimplanted. As expected, only the mice implanted with 6 million cellsgrew tumors.

We have demonstrated that human recombinant NME7_(AB) is comparable insize and sequence to NME7-X1 and to a 30-33 kDa NME7 cleavage product.We have shown that NME7_(AB) promotes cancerous growth and causes cancercells to accelerate to the highly metastatic cancer stem cell (CSC)state also called tumor initiating cells (TIC). Therefore, we concludethat NME7-X1 and an NME7 cleavage product that removes the DM10 domainalso promote cancerous growth and causes cancer cells to accelerate tothe highly metastatic cancer stem cell (CSC) state also called tumorinitiating cells (TIC). In one example, NME7_(AB) was added to cancercells in a serum-free media and in the absence of any other growthfactors or cytokines. Within 7-10 days, the cancer cells had reverted tothe highly metastatic CSCs/TICs as evidenced by more than 100-foldincrease in the expression of molecular markers such as CXCR4, which areindicators of metastatic cancer cells. In one example, T47D breastcancer cells were cultured in either standard RPMI media or in a MinimalStem Cell Media (Example 1) to which was added recombinant NME7_(AB) toa final concentration of 16 nM. After 10 days cells were collected andanalyzed by RT-PCR for expression of molecular markers of CSCs whichwere elevated by 10-200-times (FIG. 2 ). This is a specific, detailedexample of how we transformed one cancer cell type to a more metastaticstate. It is not intended that the invention be limited by these detailsas there are a range of cancer cells that are transformed in this way, arange of reagents that revert stem cells to a more naïve state that alsoprogress cancer cells to a more metastatic state and a range ofconcentrations over which the added reagents transform the cancer cells.Other types of cancer cells have required longer periods of culture inNME7_(AB) for dramatic upregulation of metastatic markers and ability toform tumors from very low numbers of cancer cells implanted. Forexample, prostate cancer cells cultured in NME7_(AB), 2i, human NME1 orbacterial NME1 that has high homology to human NME1 or human NME7 showeddramatic increase in metastatic markers after 2-3 passages.

Metastasis marker CXCR4 is particularly elevated in metastatic breastcancer cells, while CHD1 is particularly elevated in metastatic prostatecancer. Here we show that pluripotent stem cell markers such as OCT4,SOX2, NANOG, KLF2/4 and TBX3 are also up-regulated when cancer cellstransform to more metastatic cells.

DU145 prostate cancer cells were cultured similarly and those cellscultured in NME7_(AB) also showed dramatic increases in expression ofCSC markers (FIG. 3 ). In prostate cancer cells, CHD1 (aka E-cadherin)and CXCR4 were up-regulated compared to the control cancer cells, whichwere not grown in NME7_(AB), along with other pluripotent stem cellmarkers. FIG. 20A-20C shows that ovarian cancer cell lines SK-OV3, OV-90and breast cancer cell line MDA-MB all transitioned from adherent tonon-adherent floater cells and increased expression of metastaticmarkers after 72 or 144 hours in culture with NME7_(AB). Ovarian,prostate, pancreatic cancer cells and melanoma cells were also culturedin NME7_(AB) and were transformed to a more metastatic state after asfew as 3 days in culture. FIG. 21 shows that breast, ovarian, prostate,pancreatic cancer cells and melanoma cells express MUC1 and MUC1*.

Here we have shown that NME7_(AB) transforms a wide range of cancercells to a more metastatic state. We have also shown that cancer cellsexpress a naturally occurring species that is approximately the samemolecular weight as recombinant NME7_(AB) 33 kDa (FIG. 17 , FIG. 18 .FIG. 19 , and FIG. 22 and is also devoid of the DM10 domain likeNME7_(AB) and also express an alternative isoform NME7-X1 30 kDa whichis the same sequence as NME7_(AB) except is missing 33 amino acids fromthe N-terminus. A co-immunoprecipitation experiment was performed onT47D breast cancer cells, wherein the cell extracts were incubated withan antibody against the MUC1 cytoplasmic tail, Ab-5, or a controlantibody, IgG, and co-immunoprecipitated. The immunoprecipitated specieswere separated by gel electrophoresis. The gels were blotted with twodifferent commercially available anti-NME7 antibodies. Both gels showunique NME7 bands at ˜33 kDa and ˜30 kDa (FIG. 22A-22B). The gels werestripped and re-probed with an antibody against the extracellular domainof MUC1*, anti-PSMGFR (FIG. 22C-22D), which shows that the NME7 speciesand MUC1* interact. A recombinant NME7_(AB) and a recombinant NME7-X1that we made were mixed together and run on a gel, then probed with ananti-NME7 antibody, showing that the two unique NME7 species that arenaturally occurring in breast cancer cells and that interact with MUC1*are an NME7_(AB)-like species and NME7-X1 (FIG. 22E). A similarexperiment was carried out in human stem cells. FIG. 23A-23C showphotographs of Western blots of a co-immunoprecipitation experiment.Human induced pluripotent stem, iPS7, or embryonic stem, HES3, cellextracts were incubated with an antibody against the MUC1 cytoplasmictail, Ab-5, or a control antibody, IgG, and co-immunoprecipitated. Thegel was blotted with a commercially available anti-NME7 antibody B9(FIG. 23A). Both cell types show unique NME7 bands at ˜33 kDa and ˜30kDa. The gel was stripped and re-probed with an antibody against theextracellular domain of MUC1*, anti-PSMGFR (FIG. 23B), which shows thatthe NME7 species and MUC1* interact. A recombinant NME7_(AB) and arecombinant NME7-X1 that we made were mixed together and run on a gel,then probed with an anti-NME7 antibody, showing that the two unique NME7species that are naturally occurring in breast cancer cells and thatinteract with MUC1* are an NME7_(AB)-like species and NME7-X1 (FIG.23C). Because NME7_(AB) is a recombinant protein, we do not know if thenaturally occurring species may contain an extra 1-15 additional aminoacids or devoid of 1-15 additional amino acids than the recombinantNME7_(AB), yet run with the same apparent molecular weight. By“NME7_(AB)-like”, we mean an NME7 species that runs with an apparentmolecular weight of approximately 33 kDa that is able to function theway the recombinant NME7_(AB) does, in that it is able to stimulatecancer cell growth, induce transition of cancer cells to a moremetastatic state and is able to fully support pluripotent growth ofhuman stem cells.

We conclude that cancer cell lines and cancer cell populations thatexpress NME7 and lower molecular weight NME7 species contain some cancercells that are CSCs or metastatic cancer cells. These cancers can bemade more metastatic or increase the population of cells that aremetastatic by culturing the cells in NME7_(AB), NME7-X1 or lowermolecular weight NME7 species. FIG. 19 shows a Western blot of a panelof cancer cells all expressing NME7 as well as lower molecular weightspecies NME7_(AB)-like at 33 kDa and NME7-X1 at 30 kDa. FIG. 21 showsthat cancer cell lines T47D breast cancer, PC3 and DU145 prostatecancer, BT-474 breast cancer, CHL-1 and A2058 both melanoma cell linesand CAPAN-2 and PANC-1 both pancreatic cell lines all express MUC1 andMUC1*. In FIG. 21A, BT474 cells appear not to express MUC1 or MUC1*however, we previously showed (Fessler et al 2009) that when these HER2positive breast cancer cells become resistant to chemotherapy drugs,i.e. metastatic, they do so by increasing expression of MUC1* (FIG.21D). Blocking the MUC1* receptor with an anti-MUC1* Fab reversed theirresistance to Herceptin (FIG. 21E), Taxol (FIG. 21F) as well as otherchemo agents. These cancer types and other cancer types that expressNME7 and lower molecular weight NME7 species such as 33 kDa, 30 kDa canbe made more metastatic or increase the population of cells that aremetastatic by culturing the cells in NME7_(AB), NME7-X1 or lowermolecular weight NME7 species.

Conversely, the metastatic potential of these and other cancer typesthat express NME7 and lower molecular weight NME7 species such as 33 kDaor 30 kDa can be reversed by treating the cells with anti-NME7antibodies. Anti-NME7 antibodies or antibodies that bind to NME7_(AB) orNME7-X1 are administered to a patient for the treatment or prevention ofcancers including breast, prostate, ovarian, pancreatic and livercancers. Because we have shown that NME7_(AB) exerts its tumorigeniceffects by binding to and activating the MUC1* growth factor receptor,anti-NME7 antibodies will be effective against any MUC1*-positivecancers, which include but are not limited to breast, lung, liver,pancreatic, gastric colorectal, prostate, brain, melanoma, kidney andothers. Anti-NME7, anti-NME7_(AB) or anti-NME7-X1 antibodies areadministered to patients for the treatment or prevention of cancers thatare NME7_(AB), NME7_(AB)-like, or NME7-X1 positive or a MUC1* positive.

Testing Patient Cancer Cells for Effective Therapies

NME7_(AB), NME7-X1 as well as 2i and other reagents that revert stemcells to a more naïve state also induce cancer cells to transform to amore metastatic state. After treatment with any one or combination ofthese reagents, cancer cells have a higher engraftment rate and requireup to 100,000-times less cells to cause a tumor to form in a testanimal. Therefore, methods described in this disclosure can be used toenable xenografting of a patient's primary tumor cells into a testanimal.

Candidate therapeutic agents can then be tested on the recipient animal.Effective therapeutic agents identified in this way can be used to treatthe donor patient or other patients with similar cancers. In oneembodiment, a method of identifying effective therapeutics for aparticular patient or a particular type of cancer comprises the stepsof: 1) cancer cells are obtained from a cell line, a patient or apatient to whom the therapeutic being tested will be administered; 2)cancer cells are cultured in NME7_(AB), NME7-X1, human NME1, bacterialNME1 that has high homology to human NME1 or NME7, 2i, or other reagentsshown to revert stem cells to a more naïve state; 3) resultant cancercells are implanted into a test animal to which human NME7_(AB),NME7-X1, human NME1, bacterial NME1 that has high homology to human NME1or NME7, 2i, or other reagents shown to revert stem cells to a morenaïve state may also be administered or animal is transgenic for humanNME7_(AB) or NME7-X1; 4) candidate anti-cancer therapeutic agents areadministered to the animal; 5) efficacy of the therapeutic agents areassessed; and 6) effective therapeutic agent is administered to thedonor patient or to another patient with similar cancer.

Anti-NME7 Antibodies

Anti-NME7 antibodies are potent anti-cancer agents. NME7 is a growthfactor that promotes the growth of cancer cells and also promotes theirprogression to a more metastatic state or a more aggressive state. NME7and a truncated form of NME7 that is ˜33 kDa or 30 kDa have been shownto fully support cancer growth even in serum-free media devoid of anyother growth factors or cytokines. In pull-down assays, ELISAs andnanoparticle binding experiments, we have shown that the growth factorreceptor MUC1* is a binding partner of NME7 and NME7_(AB). Promotion ofthis interaction by eliminating all other growth factors or cytokinesincreased expression of cancer stem cell markers. Blocking thisinteraction even in the presence of serum, using a polyclonal antibodythat specifically binds to NME7 actively killed the cancer cells. Thus,anti-NME7 or anti-NME7_(AB) antibodies are potent anti-cancer agentsthat can be administered to a patient for the treatment or prevention ofcancers. More than 75% of all cancers are MUC1* positive. MUC1* is thetransmembrane cleavage product of MUC1 wherein most of the extracellulardomain has been shed, leaving a portion of the extracellular domain thatcontains most of the PSMGFR sequence and may contain 9-20 additionalamino acids N-terminal to the boundary of the of the PSMGFR sequence.

One aspect of the invention is a method of treating or preventing cancerin a subject, comprising administering to the subject an effectiveamount of an anti-NME7 antibody. In one instance, the anti-NME7 antibodyis able to bind to NME7_(AB). In another instance, the anti-NME7antibody is able to bind to NME7-X1. In yet another instance, theanti-NME7 antibody that is administered to a patient inhibits orprevents its binding to its target in the promotion of cancers. In onecase, the target is the extracellular domain of a cleaved MUC1. Morespecifically, the NME7 target that promotes cancer is the PSMGFR regionof the MUC1* extracellular domain. In one aspect, an effectivetherapeutic agent is one that disrupts or prevents the interactionbetween an NME7 species and MUC1* extracellular domain, consistingprimarily of the PSMGFR portion of MUC1* or the PSMGFR peptide. Agentsfor the treatment or prevention of cancers are those agents thatdirectly or indirectly inhibit the expression or function of NME7, anNME7_(AB)-like cleavage product or alternative isoform, includingNME7-X1. In one case an effective anti-cancer therapeutic agent is onethat binds to the NME7 species or disables its tumorigenic activity. Aneffective therapeutic agent for the treatment or prevention of cancersis an agent that binds to or disables NME7, an NME7_(AB)-like cleavageproduct or alternative isoform, or NME7-X1. In one aspect, thetherapeutic agents that binds to the NME7 species is an antibody. Theantibody may be polyclonal, monoclonal, bispecific, bivalent,monovalent, single chain, scFv, or an antibody mimic that may be animalin origin, human-animal chimera, humanized or human. The antibody can begenerated by inoculation or immunization with an NME7 species orfragment thereof or selected, for example from a library or a pool ofantibodies, for their ability to bind to an NME7 species, includingNME7, an NME7_(AB)-like cleavage product or alternative isoform,including NME7-X1.

Generation of Anti-NME7 Antibodies

Anti-NME7 antibodies can be generated outside of the patient such as ina host animal or in a patient. Antibodies can be generated byimmunization of NME7 or NME7 fragments or selected from a library orpool of antibodies that may be natural, synthetic, whole or antibodyfragments based on their ability to bind to desired NME7 species such asNME7_(AB) or NME7-X1. In one aspect, the antibody is generated fromimmunization with, or selected for its ability to bind to, a peptideselected from those listed in FIG. 6-9 . In another aspect, the antibodyis generated from peptides whose sequences are not identical to those ofhuman NME1 or the antibodies are selected for their ability to bind toNME7 species and their inability to bind to human NME1.

One method used to identify NME7 or NME7-X1 derived peptides that giverise to antibodies that inhibit cancer growth and inhibit transition tometastasis or peptides that are themselves inhibitory is as follows: 1)protein sequences of human NME1, human NME7, human NME7-X1 and severalbacterial or fungal NME proteins that have high sequence homology toeither human NME1 or human NME7 are aligned; 2) regions of high sequencehomology among all the NMEs are identified; 3) peptide sequences thatare unique to NME7 or NME7-X1 but are flanking the regions of highsequence homology are identified. The peptides are then synthesized andused to generate antibodies in a human or host animal. The resultantantibodies are selected for therapeutic use if: 1) they bind toNME7_(AB) or NME7-X1, but not to NME1; 2) have the ability to inhibitcancer growth; 3) have the ability to inhibit the transition of cancercells to a more metastatic state; or 4) inhibit metastasis in vivo. Insome cases, antibodies for therapeutic use are selected for theirability to disrupt binding of NME7_(AB) or NME7-X1 to the MUC1* extracellular domain, to the PSMGFR peptide or to the N-10 peptide.

Use of Anti-NME7 Antibody for Treatment of Cancer

Those antibodies that inhibit cancer growth or transition to a moremetastatic state are selected for use as anti-cancer therapeutics andmay be administered to a patient for the treatment or prevention ofcancers. Selected antibodies may be further optimized for example byengineering or making human chimera antibodies or fully humanantibodies. To demonstrate the efficacy of this approach, we selectedNME7 peptides from regions of NME7 suspected to be critical to itscancerous function. We then generated antibodies using these peptidesand then tested both the resultant antibodies as well as the immunizingpeptides for their ability to: a) inhibit cancerous growth; and b)inhibit the induced transition from cancer cells to metastatic cancercells. NME7 peptides were selected as immunizing agents for antibodyproduction and as inhibitory agents themselves (FIG. 9 , Example 7).Peptides A1 (SEQ ID NO:141), A2 (SEQ ID NO:142), B1 (SEQ ID NO:143), B2(SEQ ID NO:144) and B3 (SEQ ID NO:145), wherein A refers to the domainfrom which the peptide is derived, i.e. the NDPK A domain and the Bdenotes that the peptide is derived from the NDPK B domain (FIG. 5 ).Each peptide was used as an immunogen and injected into 2 rabbits eachfor production of polyclonal antibodies. The antibodies that wereharvested from the blood of the immunized rabbits were purified over acolumn derivatized with the immunizing peptide. The purified antibodieswere then tested for their ability to bind to human NME7. All of theresultant antibodies bound to human NME7 but not human NME1 as desired(FIG. 10A-10B, Example 8). These results show that by choosing peptideswhose sequence is found in NME7 but not exactly identical in NME1,antibodies are generated that specifically bind to NME7 but not NME1.Because NME1 has healthy function, it is in most cases desirable togenerate antibodies that do not interfere with NME1. The antibodies werealso tested for their ability to inhibit the binding of NME7 to a MUC1*extracellular domain peptide. The ELISA experiment shown in FIG. 11shows that the antibodies inhibited the binding of NME7_(AB) to a MUC1*extracellular domain peptide much more than they inhibited binding ofNME1. Recall that each of the NME7 A domain and B domain can bind to aPSMGFR peptide. Therefore, complete inhibition of NME7_(AB) binding to aPSMGFR peptide cannot be accomplished with a single antibody or peptidethat is derived from just one domain. These antibodies and theirrespective immunizing peptides also inhibited cancer cell growth (FIG.12-13 ). These antibodies also inhibited the formation of non-adherent“floater” cells that result from growing cancer cells in NME7_(AB) (FIG.14 ). As can be seen in the figure, the polyclonal antibody generated byimmunization with the B3 peptide reduced the number of metastaticfloater cells by 95%, indicating that anti-NME7 antibodies that bind tothe B3 peptide are most effective at inhibiting cancer metastasis.Similarly, the antibodies inhibited the expression of metastatic markerCXCR4 (FIG. 15A). Again, the B3 antibodies were most efficient atinhibiting expression of CXCR4; bar labeled NME7 FL (NME7 floater cells)shows 70-fold increase in CXCR4 that B3 antibody 61 decreased to 20-fold(bar labeled NME7+61 FL). In addition, the immunizing peptidesthemselves inhibited the upregulation of CXCR4 and other metastaticmarkers when T47D cancer cells were grown in NME7_(AB) or 2i.

This is but one example of selecting peptides that generate antibodiesthat inhibit the cancerous function of NME7 and NME7 species. Sequencealignment among human NME1, human NME7, human NME7-X1 and bacterial NMEproteins that had high sequence homology to human NME1 or NME7identified five regions of homology. The fact that peptides A1, A2, B1,B2 and B3 all generated antibodies that inhibited cancer growth or theirtransition to a metastatic state means that the five regions from whichthese peptides were derived are regions of NME7 that are important forits function in the promotion of cancer. Other peptides from theseregions will also give rise to anti-NME7 antibodies that will inhibitcancer growth and metastasis and are therefore potent anti-cancertherapeutics. Antibodies generated from peptides A1, A2, B1, B2 and B3were shown to inhibit cancer growth and inhibited the transition to amore metastatic state. Monoclonal antibodies generated by immunizationwith the same or similar peptides and subsequent testing of themonoclonals will identify antibodies that, after humanizing or otherengineering known to those skilled in the art, would be administered toa patient for the treatment or prevention of cancers.

In a particular experiment, the antibodies generated by immunizationwith peptides A1, A2, B1, B2 and B3, as well as the immunizing peptidesthemselves, were added to cancer cells in culture to see if the additionof the antibodies or the immunizing peptides would inhibit cancer cellgrowth. At low concentrations and added separately, the antibodies aswell as the immunizing peptides inhibited cancer cells growth (FIG. 12for one example). However, when added at higher concentrations orcombined, the antibodies as well as the immunizing peptides robustlyinhibited cancer cell growth (FIG. 13 ). The corresponding human NME7amino acid numbers of immunizing peptides A1, A2, B1, B2 and B3 are127-142, 181-191, 263-282, 287-301, 343-371, respectively, from humanfull-length NME7 having SEQ ID NO:82 or 147.

To clarify, when residue numbers of NME7 are discussed, they refer tothe residue numbers of NME7 as set forth in SEQ ID NO:82 or 147.

The antibody used in cancer growth inhibition experiments and one of theantibodies shown in FIG. 12 was generated by immunizing with NME7peptide corresponding to amino acids 100-376 of NME7 (SEQ ID NO:82 or147). To generate higher affinity and specific anti-NME7 antibodies, thefollowing steps are followed: immunize animal with a peptide containinghuman NME7 amino acids 100-376, then: 1) de-select those antibodies thatbind to human NME1; 2) select those antibodies that inhibit NME7_(AB),2i, or other NME induced transition of cancer cells to a more metastaticstate; 3) select those antibodies that inhibit the growth of cancercells; 4) select those antibodies that inhibit the growth of MUC1*positive cancer cells; 5) select those antibodies that inhibit bindingof NME7_(AB) or NME7-X1 to MUC1* extracellular domain, essentiallyinhibit binding to the PSMGFR peptide; and/or 6) select those antibodiesthat bind to one or more of the peptides listed in FIG. 9 —A1, A2, B1,B2 or B3 peptides.

Higher affinity monoclonal antibodies or monoclonal antibodies generatedfrom longer peptides may be more effective antibody therapeutics.Alternatively, combinations of anti-NME7, anti-NME7_(AB) or anti-NME7-X1antibodies are administered to a patient to increase efficacy.

Anti-NME7 Antibodies Inhibit the Transition of Cancer Cells toMetastatic Cancer Cells.

Anti-NME7 antibodies inhibit transition of cancer cells to metastaticcancer cells also called cancer stem cells (CSCs) or tumor initiatingcells (TICs). Recall that we have demonstrated that culturing a widevariety of cancer cells in the presence of NME7_(AB) causes them totransition from regular cancer cells to the metastatic CSCs or TICs.Thus, antibodies that bind to NME7, NME7_(AB) or NME7-X1 will inhibitthe progression of cancer cells to a more metastatic state.

Cancer cells transform to a more metastatic state when cultured in thepresence of agents that revert stem cells to a more naïve state. We havedemonstrated that culturing cancer cells in NME7_(AB), human NME1dimers, bacterial NME1 dimers or MEK and GSK3-beta inhibitors, called“2i”, causes the cells to become more metastatic. As the cellstransition to a more metastatic state, they become non-adherent or lessadherent and float off of the culture plate. These floating cells,“floaters” were collected separately from those that were adherent andwere shown to: a) express much higher levels of metastatic genes; and b)generated tumors when xenografted into mice at very low copy number.RT-PCR measurement of specific metastatic markers such as CXCR4 forbreast cancers, CHD1 for prostate cancer, and other pluripotent stemcell markers such as OCT4, SOX2, NANOG, KLF4 and others weredramatically over-expressed in cancer cells that were cultured inNME7_(AB) and most over-expressed in the cells that became non-adherent,called “floaters” here and in figures.

In one example, NME7_(AB) specific antibodies, generated by immunizationwith NME7-derived peptides A1, A2, B1, B2 and B3, as well as theimmunizing peptides themselves, were added into the media along witheither NME7_(AB) or 2i to determine if they inhibited the transformationof regular cancer cells to metastatic cancer stem cells. Antibodies andpeptides were separately added along with the agent that causesmetastatic transformation; in this case NME7_(AB) or the 2i inhibitorsPD0325901 and CHIR99021. NME7_(AB) and 2i were separately used to inducethe cancer cells to be transformed to a more aggressive metastaticstate. 2i was used so that it could not be argued that the antibodiesthat were added to the media simply sopped up all of the NME7_(AB) sothat the causative agent effectively was not there (Example 10).

Visual observation was independently recorded by two scientists as theexperiment progressed (FIG. 14 ). The most striking observation was thatthe antibodies and the peptides dramatically reduced the number offloater cells, which was the first indication that the antibodies andpeptides inhibit the transformation to metastatic cancer cells. Inparticular, cells to which the antibody generated from immunization withthe B3 peptide barely generated any floater cells. mRNA was extractedfrom both the floater cells, the adherent cells and the control cancercells. The amount of mRNA, which indicates cell viability and growth,was measured. Cells that were treated with antibody had much less mRNA,indicating less live dividing cells (FIG. 16 ), which confirms thatanti-NME7_(AB) antibodies inhibit cancer cell growth as well as theirtransition to a more metastatic state. RT-PCR was used to measureexpression levels of metastatic markers, including CXCR4. Treatment withthe anti-NME7 antibodies greatly reduced the amount of metastaticmarkers, such as CXCR4, indicating that the anti-NME7 antibodies orpeptides inhibit the transition to metastatic cancer (FIG. 15A-15C).These results show that antibodies that bind to NME7_(AB) can beadministered to a patient for the treatment or prevention of metastaticcancers.

Peptides Derived from NME7_(AB) or NME7-X1 Competitively Inhibit theBinding of Intact NME7_(AB) and NME7-X1 and are Anti-Cancer Agents.

In another aspect of the invention, therapeutic agents for the treatmentor prevention of cancers are peptides derived from the NME7 sequence,which are administered to a patient for the treatment or prevention ofcancers. In one aspect, the NME7-derived peptides are administered to apatient so that the peptides, which should be shorter than the entireNME7 and unable to confer the oncogenic activity of NME7, bind to thetargets of NME7 and competitively inhibit the interaction of intact NME7with its targets, wherein such interactions promote cancer. SinceNME7_(AB) is fully able to confer oncogenic activity, the sequence ofNME7_(AB) is preferred as the source for the shorter peptide(s), whereinit must be confirmed that the peptides themselves are not able topromote cancerous growth or other tumorigenic or oncogenic activity. Ina preferred embodiment, one or more peptides having the sequence of aportion of NME7_(AB) and being preferably about 12-56 amino acids inlength are administered to a patient. To increase half-life, thepeptides may be peptide mimics, such as peptides with unnatural backboneor D-form amino acids for L. In yet another case, the anti-cancertherapeutic agent is a peptide or peptide mimic wherein the peptide hasa sequence highly homologous to at least a portion of NME7, NME7_(AB),or NME7-X1 or its target the MUC1* extracellular domain, comprising thePSMGFR peptide, also called “FLR” in some cases herein.

FIG. 6 -FIG. 9 provide a listing of preferred amino acid sequences thatare predicted to inhibit NME7 binding to its cognate target. In a stillmore preferred embodiment, the peptides that are chosen foradministration to a patient suffering from cancer or at risk ofdeveloping cancer are chosen because they bind to an NME7 bindingpartner and they do not themselves confer tumorigenic activity. In a yetmore preferred embodiment, the NME7 binding partner is the extracellulardomain of MUC1*. In a still more preferred embodiment, the NME7 bindingpartner is the PSMGFR peptide.

By the term “conferring tumorigenic activity or oncogenic activity”, itis meant that the peptides themselves cannot support or promote thegrowth of cancers. Another way of testing whether or not a peptide orpeptides derived from NME7 can promote tumorigenesis is to test whetheror not the peptides can support pluripotent growth of human stem cells.NME proteins and peptides that support pluripotent human stem cellgrowth also support cancer growth. In yet another method, peptides arede-selected if they can cause somatic cells to revert to a less maturestate.

Fragments of NME7_(AB) inhibit cancer cell growth and the transition ofcancer cells to a more metastatic state. As a demonstration, NME7peptides A1, A2, B1, B2 and B3 added separately (FIG. 12 ) or incombinations (FIG. 13 ) inhibit the growth of cancer cells. In addition,NME7 peptides A1, A2, B1, B2 and B3 inhibited the transition of cancercell to a more metastatic state (FIG. 15 ).

Thus, antibodies generated by immunizing with peptides specific to NME7,and specific to NME7_(AB) or NME7-X1 will block the cancerous action ofNME7 species and will be potent anti-cancer agents. Similarly, theseresults show that the peptides specific to NME7, and specific toNME7_(AB) or NME7-X1 will block the cancerous action of NME7 species. Inone aspect of the invention, the peptides are chosen from the list shownin FIG. 6 . In one aspect of the invention the peptides are chosen fromthe list shown in FIG. 7 . In one aspect of the invention the peptidesare chosen from the list shown in FIG. 8 . In yet another aspect of theinvention the peptides are chosen from the list shown in FIG. 9 . Theseantibodies may be generated by immunizing or may be generated orselected by other means, then selected for their ability to bind toNME7, NME7_(AB), NME7-X1 or NME7 derived peptides, including but notlimited to NME7 derived peptides A1 (SEQ ID NO: 141), A2 (SEQ ID NO:142), B1 (SEQ ID NO: 143), B2 (SEQ ID NO: 144) or B3 (SEQ ID NO: 145).Such antibodies may be polyclonal, monoclonal, bispecific, bivalent,monovalent, single chain, scFv, human or humanized or may be an antibodymimic such as protein scaffolds that present recognition regions thatbind to a specific target.

Anti-NME7 antibodies for use in the treatment or prevention of cancerscan be generated by standard methods known to those skilled in the artwherein those methods are used to generate antibodies or antibody-likemolecules that recognize NME7, NME7_(AB) or a shorter form of NME7_(AB)wherein an additional 10-25 amino acids form the N-terminus are notpresent. Such antibodies may be human or humanized. Such antibodies maybe polyclonal, monoclonal, bispecific, bivalent, monovalent, singlechain, scFv, human or humanized or may be an antibody mimic such asprotein scaffolds that present recognition regions that bind to aspecific target.

Anti-NME7 antibodies that are generated by immunization with the NME7derived peptides A1 (SEQ ID NO: 141), A2 (SEQ ID NO: 142), B1 (SEQ IDNO: 143), B2 (SEQ ID NO: 144) or B3 (SEQ ID NO: 145) or antibodies thatbind to the A1, A2, B1, B2 or B3 peptides are antibodies that bind toNME7_(AB) and NME7-X1, but resist binding to NME1 which may be requiredfor the function of some healthy cells. Such antibodies inhibit thebinding of NME7_(AB) or NME7-X1 to their target receptor, MUC1*.Antibodies that bind to A1, A2, B1, B2 or B3 peptides are antibodies canbe administered to a patient diagnosed with or at risk of developing acancer or metastasis. Such antibodies may be human or humanized. Suchantibodies may be polyclonal, monoclonal, bispecific, bivalent,monovalent, single chain, scFv, or may be an antibody mimic such asprotein scaffolds that present recognition regions that bind to aspecific target.

Anti-NME7 antibodies that are generated by immunization with the B3peptide or antibodies that bind to the B3 peptide are especiallyspecific for the recognition of NME7_(AB) and NME7-X1. Such antibodiesare also very efficient at inhibiting the binding of NME7_(AB) orNME7-X1 to their target receptor, MUC1*. Antibodies that bind to the B3peptide are also exceptionally efficient at preventing, inhibiting andreversing cancer or cancer metastases. Such antibodies may be human orhumanized. Such antibodies may be polyclonal, monoclonal, bispecific,bivalent, monovalent, single chain, scFv, or may be an antibody mimicsuch as protein scaffolds that present recognition regions that bind toa specific target.

Note that the polyclonal antibody #61, which was generated in rabbitsimmunized with the B3 peptide, inhibited the transformation of cancercells to cancer stem cells as evidenced by antibody #61 blockingupregulation of metastatic marker CXCR4 (FIG. 15 ).

The B3 peptide (SEQ ID NO: 145) derived from NME7 has a Cysteine atposition 14, which complicates the generation of anti-NME7 antibodies.We mutated Cysteine 14 to Serine to make AIFGKTKIQNAVHSTDLPEDGLLEVQYFF(SEQ ID NO:169) and immunized animals to generate anti-NME7 monoclonalantibodies. The resultant antibodies bind to the native B3 sequence aswell as the B3Cys14Ser peptide. Seven (7) high affinity and specificmonoclonal antibodies were generated: 8F9A5A1, 8F9A4A3, 5F3A5D4,5D9E2B11, 5D9E10E4, 5D9G2C4, and 8H5H5G4. However, various sequencealignments showed that there are only three (3) unique sequenceantibodies: 8F9A5A1, 8F9A4A3, 8F9A4P3 and 5F3A5D4 as seen below. Boldedand underlined regions indicate CDR sequences.

HEAVY CHAIN ALIGNMENT 8F9A5A1HIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYV  608F9A4A3H VQLQQSGPELVKPGASVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYN 60 5D9E2B11HVQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGENPNNGVTNYN  605D9E10E4H VQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYN 60 5D9G2C4HVQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYN  605F3A5D4H VQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGENPNNGVTNYN 60 8H5H5G4HVQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYN  60:** ****:* *** :******:** ***:* *:****: **.*:*:* :* .* .* 8F9A5A1HDDFKGRFAFSLETSATTAYLQINNLKNEDTSTYFCARLR--GIRPGPLAYWGQGTLVTVS  1188FIA4A3H  QKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSLYVFYFDYWGQGTTLTVS 120 QKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTT---- 116QKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTTLTVS  120QKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTTLTVS  120QKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTTLTVS  120QKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTTLTVS  120:.***: :::::.*::***:::..*..**::.*:***  .   : ***** 8F9A4P3HA 119 (SEQ ID NO: 172) 8F9A4A3H S 121 (SEQ ID NO: 173) 5D9E2B11H- 116 (SEQ ID NO: 174) 5D9E10E4H S 121 (SEQ ID NO: 175) 5D9G2C4HS 121 (SEQ ID NO: 175) 5F3A5D4H S 121 (SEQ ID NO: 175) 8H5H5G4HS 121 (SEQ ID NO: 175) 8F9A4P3HVQLQQSGPELVKPGASVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYN  608F9A4A3H VQLQQSGPELVKPGASVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYN 60 5D9E2B11HVQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYN  605D9E10E4H VQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYN 60 5D9G2C4HVQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYN  605F3A5D4H VQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYN 60 8H5H5G4HVQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYN  60********:*****:********************************************* 8F9A4P3HQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSLYVFYFDYWGQGTTLTVS 1208F9A4A3H QKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSLYVFYFDYWGQGTTLTVS120 5D9E2B11HQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTT---- 1165D9E10E4H QKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTTLTVS120 5D9G2C4HQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTTLTVS 1205F3A5D4H QKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTTLTVS120 8H5H5G4HQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTTLTVS 120****************************************** ****** ****** 8F9A4P3HS 121 (SEQ ID NO: 173) 8F9A4A3H S 121 (SEQ ID NO: 173) 5D9E2B11H- 121 (SEQ ID NO: 174) 5D9E10E4H S 121 (SEQ ID NO: 175) 5D9G2C4HS 121 (SEQ ID NO: 175) 5F3A5D4H S 121 (SEQ ID NO: 175) 8H5H5G4HS 121 (SEQ ID NO: 175) LIGHT CHAIN ALIGNMENT 8F9A4P3LETTVTQSPASLSMAIGEKVTIRCITSTDIDDDMNWYQQKPGEPPKLLISEGNTLRPGVPS  608F9A4A3L DIQMTQTTSSLSASLGDRVTLSCSASQGISNYLNWYQQKPDGTVELLIFYTSSLHSGVPS 60 5D9E2B11LDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPS  605D9E10E4L DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPS 60 5D9G2C4LDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPS  605F3A5D4L DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPS 60 8H5H5G4LDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPS  608F9A5A1L EILLTQSPAIIAASPGEKVTITCSASSSV-SYMNWYQQKPGSSPKIWIYGISNLASGVPA 60 : :**: : :: : *::*** * :* .: . :**:****.  *: *  ..* ***: 8F9A4P3LRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPLTFGSGTKLEIKR 108 (SEQ ID NO: 185)8F9A4A3L RFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIK108 (SEQ ID NO: 1109) 5D9E2B11LRFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR 108 (SEQ ID NO: 186)5D9E10E4L RFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR108 (SEQ ID NO: 186) 5D9G2C4LRFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR 108 (SEQ ID NO: 186)5F3A5D4L RFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR108 (SEQ ID NO: 186) 8H5H5G4LRFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR 108 (SEQ ID NO: 186)8F9A5A1L RFSGSGSGTSFSFTINSMEAEDVATYYCQQRSSYPPTFGGGTKLEIKR108 (SEQ ID NO: 191) ***.** **.: :**..: **:* *** * .. * ***.********5D9E2B11L DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPS 60 5D9E10E4LDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPS  605D9G2C4L DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPS 60 5F3A5D4LDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPS  608H5H5G4L DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPS 60 ************************************************************5D9E2B11L RFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR108 (SEQ ID NO: 186) 5D9E10E4LRFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR 108 (SEQ ID NO: 186)5D9G2C4L RFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR108 (SEQ ID NO: 186) 5F3A5D4LRFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR 108 (SEQ ID NO: 186)8H5H5G4L RFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR108 (SEQ ID NO: 186) ************************************************8F9A4P3L ETTVTQSPASLSMAIGEKVTIRCITSTDIDDDMNWYQQKPGEPPKLLISEGNTLRPGVPS 60 8F9A5A1LEILLTQSPATIAASPGEKVTITCSASSSV-SYMNWYQQKPGSSPKIWIYGISNLASGVPA  60* :***** :: : ****** * :*:0: 0 *********. **: *  ..* ***: 8F9A4P3LRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPLTFGSGTKLEIKR  108 (SEQ ID NO: 185)8F9A5A1L RFSGSGSGTSFSFTINSMEAEDVATYYCQQRSSYPPTFGGGTKLEIKR 108 (SEQ ID NO: 191) ***.** **.* ***:.* :**** *** * .. * ***.********

Monoclonal antibodies 5D9E2B11, 5D9E10E4, 5D9G2C4, and 8H5H5G4 all havethe same sequence as 5F3A5D4, also known as 5D4. Herein, when we referto antibody 5F3A5D4, aka 5D4, it is understood that it also applies to5D9E2B11, 5D9E10E4, 5D9G2C4, and 8H5H5G4. As can be seen in FIG. 24 andFIG. 25 anti-NME7 antibodies 8F9A5A1, 8F9A4A3, and 5F3A5D4 all bind toNME7_(AB) but not to NME1. This is important because the A domain ofNME7 has high homology to NME1, which is required for normal cellfunction. For an anti-cancer therapeutic or an anti-metastasistherapeutic it will be imperative to inhibit NME7_(AB) but not NME1.

FIG. 26 , FIG. 27 and FIG. 28 show that these anti-NME7 antibodies arealso able to disrupt the binding of NME7_(AB) to the MUC1* PSMGFRpeptide and the N-10 PSMGFR peptide. As can be seen, there is not atotal displacement of NME7_(AB) from the MUC1* peptides. However, recallthat NME7_(AB) is comprised of an A domain and a B domain, each of whichare capable of binding to MUC1*. These antibodies were designed todisrupt binding of the B domain to MUC1*; the A domain of NME7_(AB)would still be able to bind to the MUC1* peptide on the plate surface.For a useful therapeutic, the antibody would only need to disrupt thebinding of one domain to MUC1* and in so doing ligand-induceddimerization and activation of MUC1* growth factor receptor would beblocked. Antibodies or antibody mimics that bind to the NME7 B3 peptideor the B3 Cys14Ser peptide (SEQ ID NO:169) are antibodies can beadministered to a patient diagnosed with or at risk of developing acancer or metastasis.

It is well known in the field that it is difficult to make cancer cellsmetastasize in an animal model. It is estimated that in a human tumoronly about 1 in 100,000 or even 1 in 1,000,000 cancer cells is able tobreak away from the tumor and implant elsewhere to initiate a metastasis[Al-Hajj et al., 2003]. Some researchers report that T47D breast cancercells injected into an immune compromised mouse will metastasize afterabout 12 weeks [Harrell et al 2006]. Other researchers report thatAsPC-1 pancreatic cancer cells will metastasize after about 4 weeks[Suzuki et al, 2013].

Here, we show that T47D breast cancer cells grown for 10 days in aserum-free media containing recombinant NME7_(AB) as the only growthfactor. It was observed that when grown in NME7_(AB), about 25% of thecancer cells began floating, stopped dividing but were still viable. PCRmeasurement showed that these “floating” cells greatly upregulatedexpression of the breast cancer metastatic factor CXCR4.

In some of the figures presented herein, these floater cells arereferred to as cancer stem cells (CSCs). Immune compromised female nu/numice were implanted with 90-day release estrogen pellets. Either 500,000T47D-wt cells or 10,000 T47D-CSCs (cancer stem cells) were injected intothe tail vein (i.v.), sub-cutaneously (s.c.), or into theintra-peritoneal space (i.p.) of the nu/nu mice. These cancer cells wereengineered to express Luciferase. To visualize the tumors or cancercells, animals are injected with Luciferin, then visualized on an IVISinstrument 10 minutes later. As can be seen in the IVIS measurements ofFIG. 33A—FIG. 33B, by Day 6 the 500,000 T47D-wt cells injected into thetail vein show no signs of live cancer cells or cancer cell engraftment.

In stark contrast, the 10,000 T47D-CSC injected into the tail vein havemetastasized. Before the Day 6 IVIS measurement, the T47D-CSC mice wereinjected with 32 nM recombinant NME7_(AB). The next day, one of the twoCSC mice was injected with a cocktail of anti-NME7 monoclonal antibodies8F9A5A1, 8F9A4A3, and 5F3A5D4 in a volume of 200 uL at a concentrationthat corresponds to 15 mgs/kg. The nearly coincident injection ofNME7_(AB) and anti-NME7 antibody likely nullified the effect of theantibody. FIG. 34 shows that by Day 10, the treated mouse is almostentirely metastatic. As can be seen in the figure, the mouse chosen fortreatment is more metastatic than the comparable T47D-CSC mouse.

That animal was again injected with the anti-NME7 antibodies on Day 10.The IVIS measurement of Day 12 (FIG. 35 ) shows that the antibodytreated mouse is beginning to clear the metastases. By Day 14 (FIG. 36 )the untreated mouse has died from rampant metastases and the treatedmouse has cleared the metastases. FIG. 37 shows the time course of IVISmeasurements for the mouse injected with 500,000 T47D-wt cells and themouse injected with T47D-CSCs that received anti-NME7 treatment untilDay 17 when antibody treatment was suspended. As can be seen, on Day 17there remained a small cluster of cancer cells, which by Day 19 hadgrown larger. By Day 21 the metastases had spread and antibody treatmentwas resumed. As is shown in the figure, after resumption of anti-NME7antibody treatment, the animal was cleared of all metastases and showsno signs of ill health.

FIG. 38 shows the IVIS time course for animals that were injectedsub-cutaneously or intra-peritoneally. Antibody injections for animalsinjected with CSCs sub-cutaneously or intra-peritoneally were alsoinjected with anti-NME7 antibodies s.c. or i.p.. In these animals,antibody injections stopped at Day 17 and did not resume. FIG. 39 -FIG.40 show that a polyclonal anti-NME7 antibody generated by immunizationwith the B3 peptide stains advanced cancers and metastatic cancers butnot normal tissues or low-grade cancers, where only 1 in 100,000 or 1 in1,000,000 cancer cells would be a metastatic cancer cells. Takentogether, these data show that anti-NME7 antibodies 8F9A5A1, 8F9A4A3,and 5F3A5D4 or 8F9A5A1, or 8F9A4A3, or 5F3A5D4 administered to a patientdiagnosed with or at risk of developing a cancer would prevent, inhibitthe formation of, or reverse cancer metastases.

In addition to treating metastatic animals with a cocktail ofanti-NME7_(AB) antibodies, we also administered monoclonalanti-NME7_(AB) antibodies individually and showed they were capable ofpreventing as well as reversing cancer metastases. In one demonstration,female nu/nu mice weighing approximately 20 g each, were implanted with90-day estrogen release pellets between 8-10 weeks of age. Cancer cellswere made metastatic by culturing for 10-15 days in a serum-free mediasupplemented with growth factor NME7_(AB). Both adherent and floatingcells show upregulation of metastatic markers and in animals are able tometastasize within 4-7 days. In this case, the floating cells wereharvested on Day 11 of in vitro culture and injected into the tail veinof the test animals. To test a prevention model, one group of animalswas injected into the tail vein, 24 hours before injection of themetastatic cancer cells, with anti-NME7_(AB) antibody 8F9A4A3 at 15mg/kg and injected thereafter with the same dosage approximately every48 hours. FIG. 42A—FIG. 42F shows photographs of female nu/nu mice,which were injected into the tail vein with 10,000 Luciferase positiveT47D metastatic breast cancer stem cells and treated with theanti-NME7_(AB) antibody 4A3 also known as 8F9A4A3. To image cancercells, the Luciferase substrate, Luciferin, is intraperitoneallyinjected 10 minutes before being photographed in IVIS instrument. FIG.42A-42C show IVIS photographs with animals face down. FIG. 42D-42F showIVIS photographs with animals face up. FIGS. 42A and 42D show controlanimals injected with phosphate buffered saline solution. FIGS. 42B and42E show a prevention model in which animals were injected withanti-NME7_(AB) antibody 4A3 24 hrs before injection of the metastaticcancer cells, then approximately every other day for a total of 12antibody injections over 22 days. FIGS. 42C and 42F show a reversalmodel in which animals were injected with anti-NME7_(AB) antibody 4A3 24hrs after injection of the metastatic cancer cells, then approximatelyevery other day for a total of 11 antibody injections over 20 days. Ascan be seen in the figure, anti-NME7_(AB) antibody 8F9A4A3 can prevent,as well as reverse an established metastasis.

Anti-NME7_(AB) antibodies 5A1 and 5D4 were also tested in a metastasisprevention model and shown to greatly inhibit cancer metastasis. FIG.43A-43F shows photographs of female nu/nu mice weighing approximately 20g each, which were injected into the tail vein with 10,000 Luciferasepositive T47D metastatic breast cancer stem cells and treated with theanti-NME7_(AB) antibodies 5A1, also known as 8F9A5A1, and 5D4, alsoknown as 5F3A5D4. To image cancer cells, the Luciferase substrate,Luciferin, is intraperitoneally injected 10 minutes before beingphotographed in IVIS instrument. FIG. 43A-43C show IVIS photographs withanimals face down. FIG. 43D-43F show IVIS photographs with animals faceup. FIGS. 43A and 43D show control animals injected with phosphatebuffered saline solution. FIGS. 43B, 43E, 43C and 43F show a preventionmodel in which animals were injected with anti-NME7_(AB) antibodies, at15 mg/kg 24 hours before injection of the metastatic cancer cells, thenapproximately every other day for a total of 12 antibody injections over22 days. Photographs were taken either at Day 24 or at Day 27.Specifically, mouse #1 in the group treated with antibody 5A1 wasphotographed at Day 27 while mouse #2 and #3 were photographed on Day 24because animals died on Day 26.

Anti-NME7_(AB) antibodies 5A1 and 5D4 were also tested in a metastasisreversal model and shown to greatly inhibit established cancermetastases. In this experiment, animals were injected on Day 0 into thetail vein with 10,000 T47D metastatic cancer cells mixed with NME7_(AB)at a final concentration of 32 nM. Further, animals were injected twice,Day 3 and Day 4, with more NME7_(AB) which our experiments have shownmake the metastasis more difficult to reverse. The first antibodyinjection was on Day7. Because the degree of metastasis in each testanimal is somewhat variable, we wanted to make certain that the apparentclearance of metastatic cancer cells was due to the anti-NME7_(AB)treatment. We therefore treated the animals with alternating high doseand low doses. As can clearly be seen in FIG. 44 , high doseanti-NME7_(AB) results in clearance of the metastasis, which if notcompletely eradicated comes back and even increases with lower dose.This experiment shows that all three anti-NME7_(AB) antibodies tested,5A1, 4A3 and 5D4, which are able to bind to the NME7-B3 peptide, inhibitcancer metastasis in a concentration dependent manner. FIG. 44A-44D showphotographs of female nu/nu mice that were injected into the tail veinwith 10,000 Luciferase positive T47D metastatic breast cancer stem cellsmixed with NME7_(AB) at a final concentration of 32 nM. Animals werethen injected into the tail vein with 32 nM NME7_(AB) before beingtreated with individual anti-NME7_(AB) antibodies. FIG. 44A showscontrol animals injected with phosphate buffered saline solution. FIG.44B shows animals treated with anti-NME7_(AB) monoclonal antibody8F9A5A1. FIG. 44C shows animals treated with anti-NME7_(AB) monoclonalantibody 8F9A4A3. FIG. 44D shows animals treated with anti-NME7_(AB)monoclonal antibody 5F3A5D4. Green arrows indicate low antibody dosage(5-7 mg/kg) over the indicated period and Red arrows indicate highdosage (15 mg/kg). As can be seen in the figure, the metastasis clearsconsiderably when antibody is administered at 15 mg/kg.

In addition to demonstrating that the anti-NME7_(AB) antibodies of theinvention can inhibit metastasis, we tested their effect on metastasisfrom a primary tumor, which would more closely mimic the physiology ofcancer metastasis. We generated T47D metastatic breast cancer cells,also known as cancer stem cells (CSCs) by culturing the cancer cells ina minimal serum-free media containing NME7_(AB) for 10-15 days. TheseT47D CSCs were then implanted sub-cutaneously into the right flank ofNSG mice into which had been implanted a 90-day estrogen release pellet.The implanted cancer cells were Luciferase positive so that afterinjection of the Luciferase substrate, Luciferin, the cancer cells emitphotons and can be photographed in an IVIS instrument to measure andlocate the implanted cancer cells. FIG. 45A-45B shows photographs offemale nu/nu mice that on Day 0 were injected sub-cutaneously into theright flank with 10,000 Luciferase positive T47D metastatic breastcancer stem cells, mixed with NME7_(AB) to a final concentration of 32nM, then mixed in a 1:1 vol:vol with Matrigel. Tumor engraftment wasallowed to progress Day 0-Day 6. Animals were then treated i.v. by tailvein injection with anti-NME7_(AB) antibodies. Control animals wereinjected with PBS. FIG. 45A shows IVIS photographs of control animals.FIG. 45B shows IVIS photographs of animals injected into tail vein witha cocktail of anti-NME7_(AB) antibodies 5A1, 4A3 and 5D4 to a totalconcentration of 15 mg/kg. Antibodies or PBS were administered 4 timesbetween Day 7 and Day 18. As can be seen in the figure, theanti-NME7_(AB) antibody treated animals show less metastases (blue dotsin whole body) than the control group. In the treated group, 2 of the 5animals have primary tumors that are larger than those in the controlgroup. This could be because the anti-NME7_(AB) antibodies prevented thespread of the cancer cells, so they remained concentrated in the primarytumor. In this experiment, PCR analysis, performed prior to injection ofthe cancer cells, showed that after 11 days in culture with NME7_(AB),the T47D breast cancer cells had upregulated CXCR4 by 109-fold, OCT4 by2-fold, NANOG by 3.5-fold and MUC1 by 2.7-fold.

In another experiment, we tested the effect of anti-NME7_(AB) antibodiesof the invention on metastasis from a primary tumor to organs thatbreast cancers typically metastasize to. Breast cancers commonlymetastasize to liver, lung, bone and brain, in that order. We generatedT47D metastatic breast cancer cells by culturing in a minimal serum-freemedia containing NME7_(AB) for 11 days. These T47D CSCs were thenimplanted sub-cutaneously into the right flank of NSG mice into whichhad been implanted a 90-day estrogen release pellet. FIG. 46A-46P showsphotographs of female nu/nu mice that on Day 0 were injectedsub-cutaneously into the right flank with 10,000 Luciferase positiveT47D metastatic breast cancer stem cells, mixed with NME7_(AB) to afinal concentration of 32 nM, then mixed in a 1:1 vol:vol with Matrigel.Tumor engraftment was allowed to progress Day 0-Day 6. Animals were thentreated i.v., by tail vein injection, with anti-NME7_(AB) antibodies.Control animals were injected with PBS. On Day 38 animals weresacrificed and livers harvested then analyzed by IVIS to detect cancercells that had metastasized to the liver. FIG. 46A-46B show whole bodyIVIS photographs of control animals that were injected with only PBS.FIG. 46C-46D show whole body IVIS photographs of control animals thatwere injected with the anti-NME7_(AB) antibody 5A1. FIG. 46E-46F showwhole body IVIS photographs of control animals that were injected withthe anti-NME7_(AB) antibody 4A3. FIG. 46G-46H show whole body IVISphotographs of control animals that were injected with theanti-NME7_(AB) antibody 5D4. FIGS. 46A, 46C, 46E, and 46G are IVISphotographs taken at Day 7 before any treatment. FIGS. 46B, 46D, 46F,and 46H are IVIS photographs taken at Day 31 after anti-NME7_(AB)antibody treatment or mock treatment. As can be seen in the figure,animals in the PBS control group show metastasis (blue dots) in thewhole body IVIS photographs, while animals treated with anti-NME7_(AB)antibodies do not. FIG. 461-46P show photographs and IVIS photographs oflivers and lung harvested from animals after sacrifice. FIGS. 46I, 46K,46M, and 46O are regular photographs. FIGS. 46J, 46L, 46N, and 46P areIVIS photographs, illuminating the cancer cells that have metastasizedthere. As can be seen in the figure, the anti-NME7_(AB) antibodiesgreatly inhibited metastasis to the liver, which is a primary site forbreast cancer metastasis. FIG. 46Q is a bar graph of the measuredphotons emitted and enumerated by IVIS instrument for livers harvestedfrom control animals versus the treated animals. As can be seen in theinserted graph of IVIS measurements, the inhibition of metastasis to theliver follows the rank order of inhibition of metastasis when cells wereinjected into the tail vein, which also matches the rank order ofpotency in being able to disrupt the NME7_(AB)-MUC1* interaction.

We performed immunofluorescent imaging of many cancer cell lines todetermine if cultured cancer cell lines express NME7_(AB). As FIGS.47A-47F and FIG. 48A-48I clearly show, each MUC1 positive cancer cellline we tested is positive for NME7_(AB) and its binding is membranous,consistent with NME7_(AB) being secreted from cancer cells whereupon itbinds to the extra cellular domain of MUC1*. FIG. 47A-47F showsphotographs of immunofluorescent experiments in which various cancercell lines are stained for the presence of NME7_(AB). FIG. 47A showsT47D breast cancer cells stained with varying concentrations ofanti-NME7_(AB) antibody 5D4. FIG. 47B shows ZR-75-1 breast cancer cells,also known as 1500s, stained with varying concentrations ofanti-NME7_(AB) antibody 5D4. FIG. 47C shows H1975 non-small cell lungcancer cells stained with varying concentrations of anti-NME7_(AB)antibody 5D4. FIG. 47D shows H292 non-small cell lung cancer cellsstained with varying concentrations of anti-NME7_(AB) antibody 5D4. FIG.47E shows HPAFII pancreatic cancer cells stained with varyingconcentrations of anti-NME7_(AB) antibody 5D4. FIG. 47F shows DU145prostate cancer cells stained with varying concentrations ofanti-NME7_(AB) antibody 5D4. As can be seen in the figure, all thecancer cell lines we tested show strong and membranous staining forNME7_(AB). The monoclonal antibody used in these experiments was 5D4. Inparallel, NME7_(AB) antibodies 5A1 and 4A3 were used to stain the samecell lines and produced the same results.

FIG. 48A-48I shows photographs of immunofluorescent experiments in whichvarious lung cancer cell lines are stained for the presence ofNME7_(AB). FIG. 48A-48C shows H1975 non-small cell lung cancer cells,which are an adenocarcinoma, stained with varying concentrations ofanti-NME7_(AB) antibody 5D4. FIG. 48A is an overlay of DAPI andanti-NME7_(AB) staining. FIG. 48B shows anti-NME7_(AB) staining alone.FIG. 48C is a magnified view of the overlay of DAPI and anti-NME7_(AB)staining. FIG. 48D-48F shows H292 non-small cell lung cancer cells,which are a mucoepidermoid pulmonary carcinoma, stained with varyingconcentrations of anti-NME7_(AB) antibody 5D4. FIG. 48D is an overlay ofDAPI and anti-NME7_(AB) staining. FIG. 48E shows anti-NME7_(AB) stainingalone. FIG. 48F is a magnified view of the overlay of DAPI andanti-NME7_(AB) staining. FIG. 48G-48I shows H358 non-small cell lungcancer cells, which are a metastatic bronchioalveolar carcinoma, stainedwith varying concentrations of anti-NME7_(AB) antibody 5D4. FIG. 48G isan overlay of DAPI and anti-NME7_(AB) staining. FIG. 48H showsanti-NME7_(AB) staining alone. FIG. 48I is a magnified view of theoverlay of DAPI and anti-NME7_(AB) staining.

In addition, culturing these cell lines in a serum-free media containingNME7_(AB) even further increased their expression of stem cell andmetastatic markers. In particular, the cells that became non-adherent,referred to here as floaters, have even higher expression of stem celland metastatic markers than their adherent counterparts. FIG. 49A-49Ishows PCR graphs of cancer cell lines, breast T47D, Lung H1975, lungH358 and pancreatic HPAFII before and after culture in NME7_(AB). FIG.49A measured breast metastatic marker CXCR4. FIG. 49B measured stem cellmarker OCT4. FIG. 49C measured metastatic marker ALDH1. FIG. 49Dmeasured stem cell marker SOX2. FIG. 49E measured stem cell markerNANOG. FIG. 49F measured marker CDH1, also known as E-cadherin. FIG. 49Gmeasured metastatic marker CD133. FIG. 49H measured stem cell markerZEB2. FIG. 49I measured stem, cancer and metastatic marker MUC1. Thefloater cells, also known as tumor spheres become able to grow anchorageindependently and show markers of metastasis that are more elevated thanthe adherent cells. Animals injected with cancer stem cells are thoseinjected with the NME7_(AB) grown floater cells. As can be seen in thefigure markers of metastasis, stem cell markers, or markers ofepithelial to mesenchymal transition (EMT) are elevated after culture inNME7_(AB), indicating a transition to a more metastatic state. FIG. 50shows Day 6 IVIS photographs of NSG mice injected into the tail veinwith either 10,000 H358 lung cancer parent cells or H358 cells after10-12 days in culture with NME7_(AB). As can be seen in the figure, theNCI-H358 lung cancer cells grown in NME7_(AB) have greatly increasedmetastatic potential compared to the parent cells, which are themselvesreportedly metastatic cells. The functional increase in metastasis in 6days from the NCI-H358 NME7_(AB) metastatic cancer stem cells from just10,000 cells is consistent with FIG. 49 , showing that H358 cellsgreatly increased expression of metastatic markers after culture inNME7_(AB).

FIG. 51 shows PCR graph of a MUC1 negative prostate cancer line PC3before and after 2 or 3 passages in culture in either dimeric NM23-H1,also known as NME1, or NME7_(AB). The graph shows the fold difference inmarkers of stem cells, cancer cells as well as metastatic markers. Ascan be seen in the figure, repeated culture in NME1 or NME7_(AB) inducesupregulation of stem, cancer and metastatic markers but also upregulatesexpression of MUC1 by 5-8 times.

Collectively, these data have demonstrated that an NME7 that is devoidof the DM10 domain is secreted by cancer cells and binds to the extracellular domain of a MUC1 that is devoid of tandem repeat domain,whereupon the NME7 dimerizes the MUC1* extra cellular domain whichresults in increased cancer cell growth and an increase in the cancercells' metastatic potential. It stands to reason that antibodies thatdisrupt the interaction between NME7_(AB) and MUC1* extra cellulardomain would inhibit cancer cell growth and would inhibit cancermetastasis. Here, we have shown that anti-NME7_(AB) antibodies thatinhibit interaction between NME7_(AB) and MUC1* extra cellular domain doin fact inhibit cancer cell growth and cancer metastasis. Therefore, itfollows that anti-NME7_(AB) antibodies can be administered to a patient,diagnosed with or at risk of developing a cancer or metastasis, for thetreatment or prevention of cancers.

Because NME1 is expressed in the cytoplasm of all cells and can belethal if knocked out, and importantly the NME1 A domain has highsequence homology to the NME7 A domain, it is critical thatanti-NME7_(AB) antibodies for therapeutic use bind to NME7_(AB) orNME7-X1, but not to NME1. In one aspect of the invention antibodies thatwould be optimal for therapeutic use were selected for their ability tobind to peptides that were unique to NME7_(AB) or NME7-X1 and were notpresent in the NME1 sequence. FIG. 6 -FIG. 9 lists NME7_(AB) uniquepeptides.

In a preferred embodiment, antibodies suitable for administration to apatient for the treatment or prevention of cancer or cancer metastasisare selected from the group of antibodies that bind to the NME7 B3peptide. In yet a more preferred embodiment, antibodies suitable foradministration to a patient for the treatment or prevention of cancer orcancer metastasis are selected from the group of antibodies that bind tothe NME7 B3 peptide, bind to NME7_(AB) but do not bind to NME1. Examplesof antibodies suitable for therapeutic use for the treatment orprevention of cancers or cancer metastasis, which have demonstrated suchanti-cancer activity and anti-metastatic activity in vitro and in vivohere, include anti-NME7 antibodies 5A1, 4A3 and 5D4. These are butexamples and other antibodies generated as described here and selectedas described here will have the same anti-cancer and anti-metastaticactivity. Such antibodies may be full antibodies or fragment thereof,including scFvs or antibody mimics wherein the variable domains of theantibody are incorporated into a protein scaffold that mimic anantibody. The antibodies may be of human or non-human species, includingmurine, camelid, llama, human or humanized and may be monoclonal,polyclonal, scFvs or fragments thereof.

Anti-NME7 antibodies for treatment or prevention of cancers ormetastases can be used in many different therapeutic formats. Forexample, any of the antibodies described herein, or a fragment thereof,can be administered to a patient as a stand-alone antibody or antibodyfragment, or attached to a toxin such as an antibody drug conjugate(ADC), or incorporated into a bi-specific antibody or incorporated intoa BiTE (bispecific T cell engager), or incorporated into a chimericantigen receptor (CAR) or engineered to be expressed by a cell that alsoexpresses a CAR. The cell may be an immune cell, a T cell, an NK cell ora stem or progenitor cell, which may then be differentiated into a Tcell or an NK cell.

Any of the antibodies described herein, or a fragment thereof, can beused as a diagnostic reagent to probe a bodily fluid, cell, tissue orbodily specimen for the presence of NME7_(AB) or NME7-X1, which would bean indicator of cancer or susceptibility to cancers. Antibodies fordiagnostic uses may be connected to an imaging agent, a nucleic acidtag, may be of any species including camelid, and can be used in wholebody applications or on a bodily fluid, such as blood, cell, or tissue,in vitro, in vivo or intra-operatively.

The selection criteria, for therapeutically useful or diagnosticallyuseful anti-NME7 antibodies, depends on the format or modality of thetherapeutic or diagnostic into which the antibody will be incorporated.If the antibody or antibody fragment is to be administered to a patientas a stand-alone agent for the treatment or prevention of cancers orcancer metastases, then the antibody is selected for its ability to: i)bind to NME7_(AB) or NME7-X1, but not to NME1; ii) bind to the PSMGFRpeptide; iii) bind to the N-10 peptide and iv) disrupt the interactionbetween NME7_(AB) or NME7-X1 and the MUC1* extra cellular domain or theinteraction between NME7_(AB) or NME7-X1 and the N-10 peptide. Theantibody may also be selected for its ability to bind to the NME7 B3peptide. This therapeutic format also encompasses a cell that has beenengineered to express a CAR and a secreted anti-NME7 antibody.

Other modalities require other selection criteria for anti-NME7antibodies. If the anti-NME7 antibody is to be incorporated into an ADC,the ADC must be internalized by the target cell to trigger killing ofthe target cell. Recall that NME7_(AB) or NME7-X1 will be bound to theextra cellular domain of MUC1*. If the antibody disrupts binding of theNME to MUC1* extra cellular domain, then the toxin-conjugated antibodywill not be internalized and the cell will not be killed. Similarly, ifthe anti-NME7 antibody is to be incorporated into a CAR or a BiTE, theinteraction between NME7_(AB) or NME7-X1 cannot be disrupted or theimmune cell will no longer be able to direct its killing agents to thecancer cell. If the anti-NME7 antibody is to be used as a diagnosticreagent, the interaction between NME7_(AB) or NME7-X1 cannot bedisrupted or antibody and associated label will be washed away.Therefore, for ADC, CAR T, or CAR-NK, BiTEs or diagnostic applications,the anti-NME7 antibody is selected for its ability to: i) bind toNME7_(AB) or NME7-X1, but not to NME1; ii) bind to the PSMGFR peptide;iii) bind to the N-10 peptide and iv) bind to NME7_(AB) or NME7-X1without disrupting the interaction with the MUC1* extra cellular domainor the interaction between NME7_(AB) or NME7-X1 and the N-10 peptide.The antibody may also be selected for its ability to bind to the NME7 B3peptide.

In one aspect of the invention, a cell is engineered to express ananti-NME7_(AB) antibody of the invention or fragment thereof. The cellmay be an immune cell, such as a T cell or NK cell or it may be a stemor progenitor cell, which may be differentiated into a more matureimmune cell such as a T cell or NK cell. In a preferred embodiment, thecell that is engineered to express an anti-NME7_(AB) antibody is alsoengineered to express a chimeric antigen receptor (CAR). In a preferredembodiment, the CAR recognizes a tumor associated antigen. In apreferred embodiment, the CAR targets MUC1*. In a more preferredembodiment, the CAR is directed to the tumor by anti-MUC1* antibodyMNC2. In another aspect of the invention, cell that is engineered toexpress a CAR is also engineered to inducibly express an anti-NME7antibody. In one example, the nucleic acid encoding an anti-NME7_(AB)antibody is inserted into the Foxp3 enhancer or promoter. In anotherexample, the anti-NME7_(AB) antibody is in an NFAT-inducible system. Inone aspect, the NFAT-inducible system incorporates NFATc1 responseelements inserted upstream of an anti-NME7_(AB) antibody sequence. Theymay be inserted into an IL-2 promoter, a Foxp3 enhancer or promoter orother suitable promoter or enhancer.

In another aspect of the invention, peptides that are unique toNME7_(AB) or NME7-X1 are incorporated into an entity used to immunize orvaccinate people against cancers or cancer metastases. In a preferredembodiment, the peptide comprises all or part of the NME7 B3 peptide,which may be the NME7 B3 peptide with Cys-14-Ser mutation.

Another aspect of the invention involves a method of generatinganti-NME7_(AB) antibodies in a host animal, where the animal isimmunized with the NME7 B3 peptide. In a preferred embodiment, the NME7B3 peptide has Cysteine 14 mutated to Serine (SEQ ID NO:169) to avoiddisulfide bond formation which inhibits NME7 specific antibodygeneration.

Another aspect of the invention involves a method of generating cellswith enhanced metastatic potential involving culturing the cells withNME7_(AB) or NME7-X1. These cells can then be used in many aspects ofdrug discovery.

Another aspect of the invention involves a cell that is engineered toexpress NME7_(AB) or NME7-X1. The NME7_(AB) or NME7-X1 may be of humansequence. Their expression may be inducible. In one aspect the cell isan egg which is then developed into an animal that may be a transgenicanimal able to express human NME7_(AB) or NME7-X1.

NME7 binds to and dimerizes the extra cellular domain of the MUC1*growth factor receptor. Tissue studies show that MUC1* increases astumor grade and metastasis increase. Here we show that NME7 expressionincreases as tumor grade and metastasis increases (FIG. 39 -FIG. 41 ).Here, we have shown that antibodies that inhibit the interaction of NME7and MUC1* inhibit tumor growth and metastases.

Other NME family members may bind to and dimerize the extra cellulardomain of the MUC1* growth factor receptor. For example, we have shownthat NME1, NME2 and NME6 can exist as dimers and that they bind to anddimerize the MUC1* extra cellular domain. NME7_(AB) and NME7-X1 have twodomains that can bind to the MUC1* extra cellular domain so as monomersthey dimerize and activate the MUC1* growth factor receptor. We have nowshown that anti-NME7 antibodies inhibit cancer and cancer metastases.Similarly, antibodies or antibody mimics that bind to these other NMEproteins may be anti-cancer or anti-metastasis therapeutics that can beadministered to a patient diagnosed with or at risk of developing acancer or a metastasis. In one aspect of the invention, antibodies thatcan be used therapeutically for the treatment of cancers or metastasesare antibodies that bind to NME1, NME2, NME3, NME4, NME5, NME6, NME7,NME8, NME9 or NME10. In one aspect of the invention, the therapeuticantibody or antibody mimic inhibits the binding of the NME protein andits cognate growth factor receptor. In one aspect of the invention, thetherapeutic antibody or antibody mimic inhibits the interaction of theNME protein with the extra cellular domain of MUC1*. In another aspectof the invention, the therapeutic antibody or antibody mimic binds to apeptide, derived from NME1, NME2, NME3, NME4, NME5, NME6, NME7, NME8,NME9 or NME10, wherein the peptide is homologous to the NME7 A1, A2, B1,B2 or B3 peptide.

Below is a sequence alignment that shows a homology and identityalignment between NME7 and other NME family members. The underlined orunderlined and bolded sequences correspond to NME7 peptides A1 (SEQ IDNO: 141), A2 (SEQ ID NO: 142), B1 (SEQ ID NO: 143), B2 (SEQ ID NO: 144)and B3 (SEQ ID NO: 145).

nucleoside diphosphate kinase 7 isoform a [Homo sapiens] (Hu_7)MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEITEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN (SEQ ID NO: 1141) NME2 Theoretical pI/Mw: 8.52/17298.04MANLERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVAMKFLRASEEHLKQHYIDLKDRPFFPGLVKYMNSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGSDSVKSAEKEISLWFKPEELVDYKSCAHDWVYE (SEQ ID NO: 19)global/global (N-W) score: 171; 26.5% identity (56.8% similar) in 155 aaoverlap (1-131:1-152)            10           20       30        40        50 7A----EKTLALIKPDAISKA--GEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPF    :.:.  ::::.....  ::::. ... :: .. .:..  :...  . ..: ..::: 2MANLERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVAMKFLRASEEHLKQHYIDLKDRPF        10        20        30        40        50        60    60        70        80        90        100      110 7AFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAA:  :......::..::     ...   . .:: .: . ..     .::. :  .  :: 2FPGLVKYMNSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPG---TIRGDFCIQVGRNII        70        80        90       100           110    120       1307A HGPDSFASAAREMELFF------------------ (SEQ ID NO: 199):: ::  :: .:. :.: 2 HGSDSVKSAEKEISLWFKPEELVDYKSCAHDWVYE (SEQ ID NO: 200)120       130       140       150global/global (N-W) score: 104; 24.4% identity (51.3% similar) in 156 aaoverlap (1-134:1-152)            10        20        30        40        50 7BNC----TCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVT      :   .:: .:..::.:.:.  ...  ::.. ::...  .. .... :  : 2MANLERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVAMKFLRASEEHLKQHYIDLKDR-P        10        20        30        40        50   60        70        80        90       100       110 7B EYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNA  .  .:  : ::: :::  .  :..:: : . : ..:  ..   :::.:. :     .: 2 FFPGLVKYMNSGPVVAMVWEGLNVVKTGRVMLGETNPADSK---PGTIRGDFCIQVGRNI60    70    80    90    100     110  120       130 7BVHCTDLPEDGLLEVQYFF------------------ (SEQ ID NO: 201) .: .:  ...  :.. .:2 IHGSDSVKSAEKEISLWFKPEELVDYKSCAHDWVYE (SEQ ID NO: 202) 120       130       140       150 >NME3 Theoretical pI/Mw: 5.96/19088.97MICLVLTIFANLFPSAYSGVNERTFLAVKPDGVQRRLVGEIVRRFERKGFKLVALKLVQASEELLREHYVELRERPFYSRLVKYMGSGPVVAMVWQGLDVVRASRALIGATDPGDATPGTIRGDFCVEVGKNVIHGSDSVESAQREIALWFREDELLCWEDSAGHWLYE (SEQ ID NO: 206)        10          20        30        40        50 7AEKTLALIKPDAISK--AGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNEL:.:.  .:::....  .:::.. ... :: .. ::... :..   . .:. . :::...: 3ERTFLAVKPDGVQRRLVGEIVRRFERKGFKLVALKLVQASEELLREHYVELRERPFYSRL       30        40        50        60        70        8060        70        80        90        100       110 7AIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASE-SIRALFGTDGIRNAAHGP.... .::..::     :..   . :.: .. :    ::.  .::. : ..  .:. :: 3VKYMGSGPVVAMVWQGLDVVRASRALIGATDPG----DATPGTIRGDFCVEVGKNVIHGS       90       100       110           120       130 120       130 7ADSFASAAREMELFF (SEQ ID NO: 203) ::  :: ::. :.: 3DSVESAQREIALWF (SEQ ID NO: 204) 140       150                             10        20        30 7BN-C--------------------TCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNM  :                    :   ::: .:.. :.:.:.  ..  ::.. :... . 3MICLVLTIFANLFPSAYSGVNERTFLAVKPDGVQRRLVGEIVRRFERKGFKLVALKLVQA        10        20        30        40        50        6040        50        60        70        80        90 7B DRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHL ..  ..: : :       :  .:  : ::: :::  :  ..... : . : .::  : 3 SEELLREHY-VELRERPFYSRLVKYMGSGPVVAMVWQGLDVVRASRALIGATDPGDAT--          70        80        90       100       110100      110       120       130 7B RPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF------------------ (SEQ ID NO: 205)  :::.:. :     .:..: .:  :..  :.  .: 3 -PGTIRGDFCVEVGKNVIHGSDSVESAQREIALWFREDELLCWEDSAGHWLYE (SEQ ID NO: 206)  120       130       140       150       160NME4 Theoretical pI/Mw: 10.30/20658.59MGGLFWRSALRGLRCGPRAPGPSLLVRHGSGGPSWTRERTLVAVKPDGVQRRLVGDVIQRFERRGFTLVGMKMLQAPESVLAEHYQDLRRKPFYPALIRYMSSGPVVAMVWEGYNVVRASRAMIGHTDSAEAAPGTIRGDFSVHISRNVIHASDSVEGAQREIQLWFQSSELVSWADGGQHSSIHPA (SEQ ID NO: 1110)29.3% identity (68.4% similar) in 133 aa overlap (1-131:56-185)        10          20        30        40        50 7AEKTLALIKPDAISK--AGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNEL:.::. .:::....  .:..:. ... :::.. .::..  ..   . . : . .::.  : 4ERTLVAVKPDGVQRRLVGDVIQRFERRGFTLVGMKMLQAPESVLAEHYQDLRRKPFYPAL   60        70        80        90       100       11060        70        80        90       100       110 7AIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPD:.....::..::     ...   . ..: ..:. :   :  .::. :..   ::. :. : 4IRYMSSGPVVAMVWEGYNVVRASRAMIGHTDSAEA---APGTIRGDFSVHISRNVIHASD  120       130       140       150          160       170 120       1307A  SFASAAREMELFF (SEQ ID NO: 207)  :  .: ::..:.: 4 SVEGAQREIQLWE (SEQ ID NO: 208)       18028.8% identity (56.8% similar) in 132 aa overlap (3-134:40-167)       10        20        30        40        50        60 7B TCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMV :   ::: .:.. :.: ... ..  :: . .:.:..  .  . : :.  .     :  .. 4 TLVAVKPDGVQRRLVGDVIQRFERRGFTLVGMKMLQAPESVLAEHYQDLRRK-PFYPALI40        50        60        70        80        90       70        80        90       100       110       120 7B TEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDL  :  ::: :::  .  :.... : . : .:   :    :::.:. :.    .:..: .: 4 RYMSSGPVVAMVWEGYNVVRASRAMIGHTDSAEAA---PGTIRGDFSVHISRNVIHASDS100       110       120       130          140       150    130 7BPEDGLLEVQYFF (SEQ ID NO: 209)  : .  :.: .: 4VEGAQREIQLWE (SEQ ID NO: 210)   160NME5 Theoretical pI/Mw: 6.08/29296.23MEISMPPPQIYVEKTLAIIKPDIVDKEEEIQDIILRSGFTIVQRRKLRLSPEQCSNFYVEKYGKMFFPNLTAYMSSGPLVAMILARHKAISYWLELLGPNNSLVAKETHPDSLRAIYGTDDLRNALHGSNDFAAAEREIRFMFPEVIVEPIPIGQAAKDYLNLHIMPTLLEGLTELCKQKPADPLFWYMCCRREHWTLRSILLVCMSGIRMSLPHCADYCSFVEGFEIWLADWLLKNNPNKPKLCHHPIVEEPY (SEQ ID NO: 1111)44.3% identity (74.8% similar) in 131 aa overlap (1-131:13-143)        10        20        30        40        50        60 7AEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQ:::::.:::: ..:  :: .:: ..::::.. . . :: ..  .:.:.. .. :: .: 5EKTLAIIKPDIVDKEEEIQDIILRSGFTIVQRRKLRLSPEQCSNFYVEKYGKMFFPNLTA      20        30        40        50        60        70    70    80    90    100    110    120 7AFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSF....::..:: . :  ::  : .:::: :: ::.    .:.::..::: .::: :: ..: 5YMSSGPLVAMILARHKAISYWLELLGPNNSLVAKETHPDSLRAIYGTDDLRNALHGSNDF   80    90    100    110    120    130        130 7AASAAREMELFF (SEQ ID NO: 211) :.: ::....: 5 AAAEREIRFMF (SEQ ID NO: 212)     140 28.0% identity (58.3% similar) in 132 aa overlap (3-134:15-143)      10        20        30        40        50        60 7BTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMV:  :.::  :..    .:   :  .:: :   . . ..  .  .:: : :     . ... 5TLAIIKPDIVDKEE--EIQDIILRSGFTIVQRRKLRLSPEQCSNFY-VEKYGKMFFPNLT    20          30        40        50         60        70      70        80        90       100       110       120 7BTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDL. : ::: ::: . ...: . . :. :: . .:..  .: .::::.:   ..::.: .. 5AYMSSGPLVAMILARHKAISYWLELLGPNNSLVAKETHPDSLRAIYGTDDLRNALHGSND       80        90       100       110       120       130      130 7BPEDGLLEVQYFF (SEQ ID NO: 213)    .  :....: 5FAAAEREIRFMF (SEQ ID NO: 214)       140NME6 Theoretical pI/Mw: 7.81 /22003.16MTQNLGSEMASILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPVCYSPEGGVHYVAGTGGLGPA (SEQ ID NO: 65)37.6% identity (68.4% similar) in 133 aa overlap (3-131:22-153)      10        20            30        40        50 7ATLALIKPDAISKAGEIIEIINKA----GFTITKLKMMMLSRKEALDFHVDHQSRPFFNEL:::::::::...   :.: ...      : :.... ..  ...   :. .:..: :...: 6TLALIKPDAVAHP-LILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRL       30         40        50        60        70        8060        70        80        90       100       110 7AIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPD..:...::: :. . . :::  :. :.::.    ::  : .:::. ::    ::..:: : 6VEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSD        90       100       110       120       130       140120       130 7A  SFASAAREMELFF (SEQ ID NO: 215)  : .::.::.  :: 6 SVVSASREIAAFF (SEQ ID NO: 216)        15029.3% identity (57.9% similar) in 133 aa overlap (3-134:22-153)      10         20        30        40        50        60 7BTCCIVKPHAVSEGL-LGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDM:  ..:: ::.. : :  . . : .  : :  :. .   . . ..::. ..:    :. . 6TLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFF-YQRL       30        40        50        60        70         80      70         80        90       100       110       120 7BVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTD:  : :::  :. . ...: . .: . ::.    :::. : ..:. :: :  .:..: .: 6VEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSD        90       100       110       120       130       140       1307B LPEDGLLEVQYFF (SEQ ID NO: 217)    ..  :.  :: 6SVVSASREIAAFF (SEQ ID NO: 218)       150NME8 Theoretical pI/Mw: 4.90/67269.94MASKKREVQLQTVINNQSLWDEMLQNKGLTVIDVYQAWCGPCRAMQPLFRKLKNELNEDEILHFAVAEADNIVTLQPFRDKCEPVFLFSVNGKITEKIQGANAPLVNKKVINLIDEERKIAAGEMARPQYPEIPLVDSDSEVSEESPCESVQELYSIAIIKPDAVISKKVLEIKRKITKAGFITEAEHKTVLTEEQVVNFYSRIADQCDFEEFVSFMTSGLSYILVVSQGSKHNPPSEETEPQTDTEPNERSEDQPEVEAQVTPGMMKNKQDSLQEYLERQHLAQLCDIEEDAANVAKFMDAFFPDFKKMKSMKLEKTLALLRPNLFHERKDDVLRIIKDEDFKILEQRQVVLSEKEAQALCKEYENEDYFNKLIENMTSGPSLALVLLRDNGLQYWKQLLGPRTVEEAIEYFPESLCAQFAMDSLPVNQLYGSDSLETAEREIQHFFPLQSTLGLIKPHATSEQREQILKIVKEAGFDLTQVKKMFLTPEQIEKIYPKVTGKDFYKDLLEMLSVGPSMVMILTKWNAVAEWRRLMGPTDPEEAKLLSPDSIRAQFGISKLKNIVHGASNAYEAKEVVNRLFEDPEEN (SEQ ID NO:1112) 36.1% identity (69.2% similar) in 133 aa overlap (1-131:316-448)        10          20       30        40        50 7AEKTLALIKPDAIS-KAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELI::::::..:. . . ....::.  : : . ....::.::: . ::::: .::.:: 8EKTLALLRPNLFHERKDDVLRIIKDEDFKILEQRQVVLSEKEAQALCKEYENEDYFNKLI  320       330       340       350       360        37060    70    80    90    100    110 7A QFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIR-NAAHGPD . .:.:: .:. .:::... ::.:::: .  :   ::. : :. :.. : .: : 8 ENMTSGPSLALVLLRDNGLQYWKQLLGPRTVEEAIEYFPESLCAQFAMDSLPVNQLYGSD  380    390    400    410    420    430  120       130 7A SFASAAREMELFF (SEQ ID NO: 219)  :. .: ::.. :: 8 SLETAEREIQHFF (SEQ ID NO: 220)        440Waterman-Eggert score: 269; 85.9 bits; E(1) < 1.1e-2133.6% identity (72.7% similar) in 128 aa overlap (1-127:451-577)        10         20        30        40        50 7AEKTLALIKPDAISKAGE-IIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELI..::.:::: : :.  : :..:...::: .:..: :.:. ..   ..    .. :...:. 8QSTLGLIKPHATSEQREQILKIVKEAGFDLTQVKKMFLTPEQIEKIYPKVTGKDFYKDLL       460       470       480       490       500       51060        70        80        90       100       110 7A QFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDS .....:: ..: . . .:. ::.::.::..   :.  . .:::: :: . ..: .:: .. 8 EMLSVGPSMVMILTKWNAVAEWRRLMGPTDPEEAKLLSPDSIRAQFGISKLKNIVHGASN       520        530       540       550       560       570 120 7A FASAAREM (SEQ ID NO: 221)   :  :.:. 8  -AYEAKEV (SEQ ID NO: 222)Waterman-Eggert score: 119; 40.4 bits; E(1) < 5.3e-0833.8% identity (73.8% similar) in 65 aa overlap (3-65:156-220)       10         20        30        40        50        60 7ATLALIKPDAI--SKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQ..:.:::::.  .:. :: . :.:::: :   . .:........:.    .. :.:... 8SIAIIKPDAVISKKVLEIKRKITKAGFIIEAEHKTVLTEEQVVNFYSRIADQCDFEEFVS  160       170      180        190       200       210 7AFITTG (SEQ ID NO: 223) :.:.: 8 FMTSG (SEQ ID NO: 224)  22033.6% identity (65.5% similar) in 116 aa overlap (3-118:453-566)      10        20        30        40        50        60 7BTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMV:  ..::::.::    .::  ...:::... .. . .   ..:..:    :    :.:.. 8TLGLIKPHATSEQRE-QILKIVKEAGFDLTQVKKMFLTPEQIEKIYPKVTGK-DFYKDLL     460        470       480       490       500        510      70        80        90       100       110 7BTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVH (SEQ ID NO: 225)  .  :: ..: . . ::.  .:.. ::.::: :. : : ..:: :: .:..: :: 8EMLSVGPSMVMILTKWNAVAEWRRLMGPTDPEEAKLLSPDSIRAQFGISKLKNIVH (SEQ IDNO: 226)       520       530       540       550       560Waterman-Eggert score: 128; 41.3 bits; E(1) < 2.9e-0823.3% identity (60.3% similar) in 116 aa overlap (20-134:334-448)20        30        40        50        60        70 7B ILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNN .:  :.:  :.:  ...  ... ... . . :..    .. .. .: ::: .:. . ..: 8 VLRIIKDEDFKILEQRQVVLSEKEAQALCKEYENE-DYFNKLIENMTSGPSLALVLLRDN     340       350       360        370       380       39080        90       100       110        120       130 7B ATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQ-NAVHCTDLPEDGLLEVQYFF (SEQ ID NO: 227) . . .... ::   : : .  : .: : :.  ..  : .. .:  : .  :.:.:: 8 GLQYWKQLLGPRTVEEAIEYFPESLCAQFAMDSLPVNQLYGSDSLETAEREIQHFF (SEQ ID NO: 228)      400       410       420       430       440Waterman-Eggert score: 76; 26.4 bits; E(1) < 0.0008823.4% identity (46.8% similar) in 111 aa overlap (6-105:159-268)    10        20        30        40        50        60 7B IVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEM :.:: ::    . .:   :  ::: : : .   . . .: .::        .....:. : 8 IIKPDAVISKKVLEIKRKITKAGFIIEAEHKTVLTEEQVVNFYSRIADQC-DFEEFVSFM160       170       180       190       200        210   70        80        90                  100 7BYSGPCVAMEIQQNNATKTFREFCGPA-----------DPEIARHLRPGTLR (SEQ ID NO: 229) ::    . ..:..  .   :   :            .::.  .. :: .. 8TSGLSYILVVSQGSKHNPPSEETEPQTDTEPNERSEDQPEVEAQVTPGMMK (SEQ ID NO: 230)220       230       240       250       260 NME9MLSSKGLTVVDVYQGWCGPCKPVVSLFQKMRIEVGLDLLHFALAEADRLDVLEKYRGKCEPTFLFYAIKDEALSDEDECVSHGKNNGEDEDMVSSERTCTLAIIKPDAVAHGKTDEIIMKIQEAGFEILTNEERTMTEAEVRLFYQHKAGESPSSVRHRNALQCRPWKPGQRRC (SEQ ID NO: 231)41.3% identity (67.4% similar) in 46 aa overlap (3-46:100-145)      10           20        30        40 7A TLALIKPDAIS--KAGEIIEIINKAGFTITKLKMMMLSRKEALDFH :::.:::::..  :. :::  :..::: :   .   ... :.  :. 9 TLAIIKPDAVAHGKTDEIIMKIQEAGFEILTNEERTMTEAEVRLFY100      110       120       130       140 >--Waterman-Eggert score: 30; 13.5 bits; E(1) < 0.8528.6% identity (71.4% similar) in 14 aa overlap (69-82:100-113) 70        80  AMEILRDDAICEWK (SEQ ID NO: 232)  .. :.. ::. . : TLAIIKPDAVAHGK (SEQ ID NO: 233) 100      110Waterman-Eggert score: 29; 13.2 bits; E(1) < 0.9125.8% identity (74.2% similar) in 31 aa overlap (12-42:121-149)       20        30        40 7AISKAGEIIEIINKAGFTITKLKMMMLSRKEA (SEQ ID NO: 234):..::  .::...   :.:. .. .. ...: 9IQEAG--FEILTNEERTMTEAEVRLFYQHKA (SEQ ID NO: 235)          130       14039.6% identity (69.8% similar) in 53 aa overlap (1-53:98-150)       10         20        30        40        50 7ANCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKG (SEQ ID NO: 236).::  :.:: ::..:   .:.: :..::::: . .  .: ...:. ::.   : 9TCTLAIIKPDAVAHGKTDEIIMKIQEAGFEILTNEERTMTEAEVRLFYQHKAG (SEQ ID NO: 237)100       110       120       130       140      150NME10 NP_008846.2 protein XRP2 [Homo sapiens]MGCFFSKRRKADKESRPENEEERPKQYSWDQREKVDPKDYMFSGLKDETVGRLPGTVAGQQFLIQDCENCNIYIFDHSATVTIDDCTNCIIFLGPVKGSVFFRNCRDCKCTLACQQFRVRDCRKLEVFLCCATQPIIESSSNIKFGCFQWYYPELAFQFKDAGLSIFNNTWSNIHDFTPVSGELNWSLLPEDAVVQDYVPIPTTEELKAVRVSTEANRSIVPISRGQRQKSSDESCLVVLFAGDYTIANARKLIDEMVGKGFFLVQTKEVSMKAEDAQRVFREKAPDFLPLLNKGPVIALEFNGDGAVEVCQLIVNEIFNGTKMFVSESKETASGDVDSFYNFADIQMGI (SEQID NO: 238)23.58 identity (66.28 similar) in 68 aa overlap (11-78:246-308)        20        30        40        50        60        70 7AAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAM.:..: ..:. .   :: ... : . .. ..:   .: ... :   ... ... ::.::. 10TIANARKLIDEMVGKGFFLVQTKEVSMKAEDAQ--RVFREKAP---DFLPLLNKGPVIAL  250       260       270         280          290       300 7AEILRDDAI (SEQ ID NO: 239) :.  : :. 10 EFNGDGAV (SEQ ID NO: 240)Waterman-Eggert score: 35; 15.1 bits; E(1) < 0.7328.9% identity (57.8% similar) in 45 aa overlap (66-108:200-244)    70         80        90       100 7A PIIAMEILRDDAIC-EWKRLLGPANSGVARTDASES-IRALFGTD (SEQ ID NO: 241) :: . : :.   .  : .: . : . :  . ...:: . .::. : 10 PIPTTEELKAVRVSTEANRSIVPISRGQRQKSSDESCLVVLFAGD (SEQ ID NO: 242)200      210       220       230       240Waterman-Eggert score: 33; 14.4 bits; E(1) < 0.8714.7% identity (52.0% similar) in 75 aa overlap (7-80:35-109)  10        20        30        40        50        60 7AIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQ-FITTG. :     .:   : ...   :..  .... . ..   .  ::..   ...  . .:  : 10VDPKDYMFSGLKDETVGRLPGTVAGQQFLIQDCENCNIYIFDHSATVTIDDCTNCIIFLG    40        50        60        70        80        90     70       807A PIIAMEILRDDAICE (SEQ ID NO: 243) :. .  ..:.   :. 10PVKGSVFFRNCRDCK (SEQ ID NO: 244)    100Waterman-Eggert score: 45; 17.5 bits; E (1) < 0.2221.6% identity (58.8% similar) in 51 aa overlap (4-50:130-180)        10        20        30          40        50 7B CCIVKP--HAVSEGLLGKILMAIRDAGFEI--SAMQMFNMDRVNVEEFYEV (SEQ ID NO: 245) :: ..:  .. :.  .: .     . .:..  .....::    :...:  : 10 CCATQPIIESSSNIKFGCFQWYYPELAFQFKDAGLSIFNNTWSNIHDFTPV (SEQ ID NO: 246)130      140       150       160       170       180

As an example, antibodies or antibody mimics that bind to the NME7homologous peptides (“homologous peptides”) in particular homologous toA1, A2, B1, B2 or B3 peptides, may be administered to a patientdiagnosed with or at risk of developing a cancer or cancer metastasis.

Homologous Peptides to A1, A2, B1, B2 or B3 Peptides

Homologous peptides to A1, A2, B1, B2 or B3 peptides may include withoutlimitation the following:

NME2A1 (amino acids) (SEQ ID NO: 247) RASEEHLKQHYIDLKD NME2A2(amino acids) (SEQ ID NO: 248) PADSKPGT NME2B1 (amino acids)(SEQ ID NO: 249) QKGFRLVAMKFLRASEEHLK NME2B2 (amino acids)(SEQ ID NO: 250) IDLKDRPFPGLVKY NME2B3 (amino acids) (SEQ ID NO: 251)GDFCIQVGRNIIHGSDSVKSAEKEISLWF NME3A1 (amino acids) (SEQ ID NO: 252)QASEELLREHYVELRE NME3A1 (amino acids) (SEQ ID NO: 253) PGDATPGT NME3B1(amino acids) (SEQ ID NO: 254) RKGFKLVALKLVQASEELLR NME3B2 (amino acids)(SEQ ID NO: 255) VELRERPFYSRLVKY NME3B3 (amino acids) (SEQ ID NO: 256)GDFCVEVGKNVIHGSDSVESAQREIALWF NME4A1 (amino acids) (SEQ ID NO: 257)QAPESVLAEHYQDLRR NME4A2 (amino acids) (SEQ ID NO: 258) SAEAAPGT NME4B1(amino acids) (SEQ ID NO: 259) RRGFTLVGMKMLQAPESVLA NME4B2 (amino acids)(SEQ ID NO: 260) QDLRRKPFYPALIRY NME4B3 (amino acids) (SEQ ID NO: 261)GDFSVHISRNVIHASDSVEGAQREIQLWF NME5A1 (amino acids) (SEQ ID NO: 262)RLSPEQCSNFYVEKYG NME5A2 (amino acids) (SEQ ID NO: 263) SLVAKETHPDSNME5B1 (amino acids) (SEQ ID NO: 264) RSGFTIVQRRKLRLSPEQCS NME5B2(amino acids) (SEQ ID NO: 265) VEKYGKMFFPNLTAY NME5B3 (amino acids)(SEQ ID NO: 266) AIYGTDDLRNALHGSNDFAAAEREIRFMF NME6A1 (amino acids)(SEQ ID NO: 267) LWRKEDCQRFYREHEG NME6A2 (amino acids) (SEQ ID NO: 268)VFRARHVAPDS NME6B1 (amino acids) (SEQ ID NO: 269) SNKFLIVRMRELLWRKEDCQNME6B2 (amino acids) (SEQ ID NO: 270) REHEGRFFYQRLVEF NME6B3(amino acids) (SEQ ID NO: 271) GSFGLTDTRNTTHGSDSVVSASREIAAFF NME8A1(amino acids) (SEQ ID NO: 272) VLSEKEAQALCKEYEN NME8A2 (amino acids)(SEQ ID NO: 273) VEEAIEYFPES NME8A3 (amino acids) (SEQ ID NO: 274)FLTPEQIEKIYPKVTG NME8A4 (amino acids) (SEQ ID NO: 275) PEEAKLLSPDSNME8A5 (amino acids) (SEQ ID NO: 276) VLTEEQVVNFYSRIAD NME8B1(amino acids) (SEQ ID NO: 277) EAGFDLTQVKKMFLTPEQIE NME8B2 (amino acids)(SEQ ID NO: 278) PKVTGKDFYKDLLEM NME8B3 (amino acids) (SEQ ID NO: 279)AQFGISKLKNIVH NME8B4 (amino acids) (SEQ ID NO: 280) DEDFKILEQRQVVLSEKEAQNME8B5 (amino acids) (SEQ ID NO: 281) KEYENEDYFNKLIEN NME8B6(amino acids) (SEQ ID NO: 282) AQFAMDSLPVNQLYGSDSLETAEREIQHFF NME8B7(amino acids) (SEQ ID NO: 283) KAGFIIEAEHKTVLTEEQVV NME8B8 (amino acids)(SEQ ID NO: 284) SRIADQCDFEEFVSF NME9A1 (amino acids) (SEQ ID NO: 285)TMTEAEVRLFY NME9B1 (amino acids) (SEQ ID NO: 286) EAGFEILTNEERTMTEAEVRNME10A1 (amino acids) (SEQ ID NO: 287) SMKAEDAQRVFREK NME10A2(amino acids) (SEQ ID NO: 288) GQRQKSSDES NME10A3 (amino acids)(SEQ ID NO: 289) IQDCENCNIYIFDHSA NME10B1 (SEQ ID NO: 290)ELAFQFKDAGLSIFNNTWSNIH

In some cases, peptides derived from other NME proteins can be made morehomologous to NME7 A1, A2, B1, B2 or B3 peptides by shifting the frameor extending the NME7 peptides such that the extended peptides are morehomologous to the NME7 peptides that gave rise to antibodies thatinhibit cancer or cancer metastases. As another example, antibodies orantibody mimics that bind to the NME7 homologous extended peptides(“extended peptides”) may be administered to a patient diagnosed with orat risk of developing a cancer or cancer metastasis.

Homologous Extended Peptides to A1, A2, B1, B2 or B3 Peptides

Homologous peptides to A1, A2, B1, B2 or B3 peptides that are extendedpeptides may include without limitation the following:

NME2A1 (amino acids) (SEQ ID NO: 291) RASEEHLKQHYIDLKDRPFFPGL NME2A2(amino acids) (SEQ ID NO: 292) LGETNPADSKPGTIRGDF NME2B1 (amino acids)(SEQ ID NO: 293) GLVGEIIKRFEQKGFRLVAMKFLRASEEHLKQHY NME2B2 (amino acids)(SEQ ID NO: 294) YIDLKDRPFFPGLVKYMNSGPVVAM NME2B3 (amino acids)(SEQ ID NO: 295) PGTIRGDFCIQVGRNIIHGSDSVKSAEKEISLWF NME3A1 (amino acids)(SEQ ID NO: 296) LKLVQASEELLREHYVELRERPFYSRL NME3A1 (amino acids)(SEQ ID NO: 297) LIGATDPGDATPGTIRGDF NME3B1 (amino acids)(SEQ ID NO: 298) LVGEIVRRFERKGFKLVALKLVQASEELLRE NME3B2 (amino acids)(SEQ ID NO: 299) EHY-VELRERPFYSRLVKYMGSGPVVAM NME3B3 (amino acids)(SEQ ID NO: 300) PGTIRGDFCVEVGKNVIHGSDSVESAQREIALWF NME4A1 (amino acids)(SEQ ID NO: 301) GFTLVGMKMLQAPESVLAEHYQDLRRKPF NME4A2 (amino acids)(SEQ ID NO: 302) GHTDSAEAAPGTIRGDF NME4B1 (amino acids) (SEQ ID NO: 303)LVGDVIQRFERRGFTLVGMKMLQAPESVLAEHY NME4B2 (amino acids) (SEQ ID NO: 304)EHYQDLRRKPFYPALIRYMSSGPVVAM NME 4B3 (amino acids) (SEQ ID NO: 305)PGTIRGDFSVHISRNVIHASDS VEGAQREIQLWF NME5A1 (amino acids)(SEQ ID NO: 306) GFTIVQRRKLRLSPEQCSNFYVEKYGKMFF NME5A2 (amino acids)(SEQ ID NO: 307) LLGPNNSLVAKETHPDSLRAIYGTD NME5B1 (amino acids)(SEQ ID NO: 308) IQDIILRSGFTIVQRRKLRLSPEQCSNFY NME5B2 (amino acids)(SEQ ID NO: 309) FYVEKYGKMFFPNLTAYMSSGPLVAM NME5B3 (amino acids)(SEQ ID NO: 310) PDSLRAIYGTDDLRNALHGSNDFAAAEREIRFMF NME6A1 (amino acids)(SEQ ID NO: 311) FLIVRMRELLWRKEDCQRFYREHEGRFFYQRL NME6A2 (amino acids)(SEQ ID NO: 312) LMGPTRVFRARHVAPDSIRGSFG NME6B1 (amino acids)(SEQ ID NO: 313) ILSNKFLIVRMRELLWRKEDCQRFY NME6B2 (amino acids)(SEQ ID NO: 314) FYREHEGRFFYQRLVEFMASGPIRA NME6B3 (amino acids)(SEQ ID NO: 315) ARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFF NME8A1(amino acids) (SEQ ID NO: 316) FKILEQRQVVLSEKEAQALCKEYENEDYFNKLI NME8A2(amino acids) (SEQ ID NO: 317) WKQLLGPRTVEEAIEYFPESLCAQFAMD NME8A3(amino acids) (SEQ ID NO: 318) AGFDLTQVKKMFLTPEQIEKIYPKVTGKDFYKDL NME8A4(amino acids) (SEQ ID NO: 319) EWRRLMGPTDPEEAKLLSPDSIRAQFG NME8A5(amino acids) (SEQ ID NO: 320) KAGFIIEAEHKTVLTEEQVVNFYSRIADQCDFEE NME8B1(amino acids) (SEQ ID NO: 321) ILKIVKEAGFDLTQVKKMFLTPEQIEKIY NME8B2(amino acids) (SEQ ID NO: 322) YPKVTGKDFYKDLLEMLSVGP NME8B3(amino acids) (SEQ ID NO: 323) DPEEAKLLSPDSIRAQFGISKLKNIVH NME8B4(amino acids) (SEQ ID NO: 324) LRIIKDEDFKILEQRQVVLSEKEAQ NME8B5(amino acids) (SEQ ID NO: 325) KEYENE-DYFNKLIENMTSGPSLA NME8B6(amino acids) (SEQ ID NO: 326) PESLCAQFAMDSLPVNQLYGSDSLETAEREIQHFFNME8B7 (amino acids) (SEQ ID NO: 327) IKRKITKAGFIIEAEHKTVLTEEQVVNFYNME8B8 (amino acids) (SEQ ID NO: 328) FYSRIADQCDFEEFVSFMTSG NME9A1(amino acids) (SEQ ID NO: 329) AGFEILTNEERTMTEAEVRLFY NME9B1(amino acids) (SEQ ID NO: 330) IIMKIQEAGFEILTNEERTMTEAEVRLFY NME10A1(amino acids) (SEQ ID NO: 331) GFFLVQTKEVSMKAEDAQRVFREKAP NME10A2(amino acids) (SEQ ID NO: 332) EANRSIVPISRGQRQKSSDESCLVVLFAGD NME10A3(amino acids) (SEQ ID NO: 333) IQDCENCNIYIFDHSA NME10B1 (SEQ ID NO: 334)ELAFQFKDAGLSIFNNTWSNIHDFTPVDCT

Some NME proteins exert a function that is necessary for normal cellgrowth or development. For example, NME1 is thought to be required fornormal cell function. Other NME proteins have catalytic domains whosefunction is required in normal cells or tissues. In these cases,therapeutic antibodies can be selected based on their ability to bind tothe targeted, cancer associated NME, but not to a non-targeted NME. Forexample, the anti-NME7 antibodies presented here, 8F9A5A1, 8F9A4A3, and5F3A5D4, were selected for their ability to bind to NME7_(AB) but not toNME1; they were further selected based on their ability to inhibitcancer and cancer metastases.

In another aspect of the invention, anti-NME7 antibodies, antibodyfragments, for example scFvs, or fragments of antibody mimics areincorporated into chimeric antigen receptors (CARs) which are engineeredto be expressed in immune cells. The immune cell can be engineered toexpress an anti-NME7 CAR, an anti-MUC1* CAR, or both. One of the CARsmay be expressed off of an inducible promoter. Alternatively, an immunecell may be engineered to express a CAR such as an anti-MUC1* CAR and aninducible anti-NME7 antibody or antibody fragment. In some instances theinducible promoter may contain NFAT response elements. In one aspect,these engineered species are expressed in T cells, NK cells or dendriticcells. The immune cells may be obtained from the patient or from adonor. In some cases, immune molecules such as MHCs, checkpointinhibitors or receptors for checkpoint inhibitors are mutated or cutout, for example using CrisPR or CrisPR-like technology. In anotheraspect, ITAM molecules, Fos, or Jun are mutated or genetically excisedvia Talens, Sleeping Beauty, CrisPR or CrisPR-like technologies inpatient or donor derived immune cells.

In one aspect of the invention, the anti-NME7 antibodies or antibodymimics for use in CAR T format are chosen from among the group ofantibodies or antibody mimics that are specific for NME7 but do notdisrupt the binding of NME7 to the extra cellular domain of MUC1*. Inthis way, the anti-NME7 antibody or antibody mimic that targets the CARTto the tumor will not simply pluck the ligand from the receptor,whereupon the T cell would be unable to inject the target cancer cellwith Granzyme B. Such antibodies or antibody mimics are generated byimmunizing an animal with an NME7 peptide, such as NME7 peptides A1, A2,B1, B2 or B3 or selected by virtue of their ability to bind to NME7peptides A1, A2, B1, B2 or B3. Antibodies or antibody mimics can bescreened for their ability to specifically bind to NME7, but not to NME1or NME2, and also for their inability to disrupt binding between NME7and MUC1* extra cellular domain. For example, in an ELISA setup, thePSMGFR peptide is immobilized to the surface. A labeled NME7_(AB) isallowed to bind to the surface immobilized MUC1* extra cellular domain,and detection of the NME7_(AB) label is measured in the presence orabsence of the test antibody or antibody mimic. In one aspect of theinvention, an antibody that does not diminish binding between NME7_(AB)and surface-immobilized MUC1* extra cellular domain peptide is selectedas an antibody that is incorporated into a CAR and engineered to beexpressed in an immune cell and then administered to a patient for thetreatment or prevention of cancer or cancer metastases.

In one aspect of the invention, an anti-NME7 antibody or fragmentthereof is administered to a patient diagnosed with or at risk ofdeveloping a cancer or cancer metastasis. In one aspect, the anti-NME7antibody or antibody fragment binds to an NME peptide discussed above inparticular under sections “Homologous peptides to A1, A2, B1, B2 or B3peptides” and the “Homologous extended peptides to A1, A2, B1, B2 or B3peptides”.

In another aspect, the antibody, antibody fragment or antibody mimicbinds to an NME7 derived peptide chosen from among A1, A2, B1, B2 or B3(SEQ ID NOS: 141-145). In yet another aspect, the antibody, antibodyfragment or antibody mimic binds to an NME7 peptide comprising most orall of the B3 peptide. In one aspect of the invention, the anti-NME7antibody, antibody fragment or antibody mimic comprises sequencesderived from the variable domains of anti-NME7 antibodies 8F9A4A3(“4A3”) (SEQ ID NOS:1001-1015), 8F9A5A1 (“5A1”) (SEQ ID NOS: 1016-1030),or 8H5H5G4 (“5G4”) (SEQ ID NOS: 1031-1045) shown below.

Anti-NME7 B3 peptide monoclonal antibodiesMonoclonal antibody 8F9A4A3 “4A3” Heavy chain variable region sequenceGaggtccagctgcaacagtctggacctgaactggtgaagcctggggcttcagtgaagatatcctgcaagacttctggaaacacattcactgaatacaccatgcactgggtgaagcagagccatggaaagagccttgagtggattggaggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaagacggtactaccatagtctctacgtgttttactttgactactggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 1138)Translated protein, wherein the underlined sequence is the complementaritydetermining region (CDR):EVQLQQSGPELVKPGASVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSLYVFYFDYWGQGTTLTVSS (SEQ ID NO: 1139)Mouse 8F9A4A3 heavy chain variable domain framework 1 (FR1) sequencegaggtccagctgcaacagtctggacctgaactggtgaagcctggggcttcagtgaagatatcctgcaagacttctgga (SEQ ID NO: 1113) EVQLQQSGPELVKPGASVKISCKTSG (SEQ ID NO: 1114)Heavy chain variable region CDR1:aacacattcactgaatacaccatgcac (SEQ ID NO: 1115) NTFTEYTMH (SEQ ID NO: 388)Mouse 8F9A4A3 heavy chain variable domain framework 2 (FR2) sequencetgggtgaagcagagccatggaaagagccttgagtggattgga (SEQ ID NO: 1116)WVKQSHGKSLEWIG (SEQ ID NO: 1117)Mouse 8F9A4A3 Heavy chain variable region CDR2:ggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggc (SEQ ID NO: 1118)GFNPNNGVTNYNQKFKG (SEQ ID NO: 389)Mouse 4A3 heavy chain variable domain framework 3 (FR3) sequenceaaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaaga (SEQ ID NO: 1119)KATLTVDKSSSTAYMELRSLTSEDSAVYYCAR (SEQ ID NO: 1120)Mouse 8F9A4A3 Heavy chain variable region CDR3:cggtactaccatagtctctacgtgttttactttgactac (SEQ ID NO: 1121)RYYHSLYVFYFDY (SEQ ID NO: 390)Mouse 8F9A4A3 Light chain variable region sequencegatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccctcagttgcagtgcaagtcagggcattagcaattatttaaactggtatcagcagaaaccagatggaactgttgaactcctgatcttttacacatcaagtttacactcaggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagcaacctggaacctgaagatattgccacttactattgtcagcagtatagtaagcttccttacacgttcggaggggggaccaagctggaaataaaa (SEQ ID NO: 1136)Translated protein, wherein the underlined sequence is the complementaritydetermining region (CDR):DIQMTQTTSSLSASLGDRVTLSCSASQGISNYLNWYQQKPDGTVELLIFYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIK(SEQ ID NO: 1109)Mouse 8F9A4A3 light chain variable domain framework 1 (FR1) sequencegatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccctcagttgc (SEQ ID NO: 1122) DIQMTQTTSSLSASLGDRVTLSC (SEQ ID NO: 1123)Mouse 8F9A4A3 Light chain variable region CDR 1:agtgcaagtcagggcattagcaattatttaaac (SEQ ID NO: 1124)SASQGISNYLN (SEQ ID NO: 393)Mouse 4A3 light chain variable domain framework 2 (FR2) sequencetggtatcagcagaaaccagatggaactgttgaactcctgatcttt (SEQ ID NO: 1125)WYQQKPDGTVELLIF (SEQ ID NO: 1126)Mouse 8F9A4A3 Light chain variable region CDR2:tacacatcaagtttacactca (SEQ ID NO: 1127) YTSSLHS (SEQ ID NO: 394)Mouse 8F9A4A3 light chain variable domain framework 3 (FR3) sequenceggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagcaacctggaacctgaagatattgccacttactattgt (SEQ ID NO: 1128)GVPSRFSGSGSGTDYSLTISNLEPEDIATYYC (SEQ ID NO: 1129)Mouse 8F9A4A3 Light chain variable region CDR3:Cagcagtatagtaagcttccttacacg (SEQ ID NO: 1130) QQYSKLPYT (SEQ ID NO: 395)Humanized 8F9A4A3 H-ori heavy chain variable domain sequencecaggttcagctggttcagtctggtgcagaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggacttgaatggatgggcggcttcaaccccaacaacggcgtgaccaactacaaccagaaattcaagggccgcgtgaccatgaccgaggacacaagcacagacaccgcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcctgtacgtgttctacttcgactactggggccagggcaccctggtcacagtttcttct (SEQ ID NO: 1131)QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWMGGFNPNNGVTNYNQKFKGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS (SEQ ID NO: 1004)Humanized 8F9A4A3 H-1.46 heavy chain variable domain sequencecaagtgcagctggtgcagagcggcgccgaggtgaagaaacctggcgccagcgtgaaagtgtcctgcaaggccagcggcaatacattcaccgagtacacaatgcactgggtcagacaggcccccggccagggcctggaatggatcggcggatttaaccccaacaacggcgtgacaaactacaaccagaagttcaagggcaaggtgaccatcacaagagacaccagcagcagcaccgtgtacatggaactgtcttctctgcggagcgaggataccgccgtgtactattgtgccagacggtactaccacagcctgtacgtgttctacttcgactactggggacagggcaccctggttaccgtgtcctct (SEQ ID NO: 1101)QVQLVQSGAEVKKPGASVKVSCKASGNTFTEYTMHWVRQAPGQGLEWIGGFNPNNGVTNYNQKFKGKVTITRDTSSSTVYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS (SEQ ID NO: 1102)Humanized 8F9A4A3 H-3.15 heavy chain variable domain sequencegaggtgcagctggtggaaagcggcggcggcctggttaagcctggcggatctctgagactgagctgtgccgcttctggcaataccttcaccgagtacaccatgcactgggtgcggcaggcccctggaaaaggcctggaatggatcggcggatttaaccccaacaacggcgtgacaaattacaaccagaaattcaagggcaagttcaccatcacaagagataagagcaagaacaccctgtacctgcaaatgaacagcctgaagtccgaggacaccgccgtgtactactgcgccagacggtactaccacagcctctatgtgttctacttcgactactggggccagggcacactggtcaccgtgtccagc (SEQ ID NO: 1132)EVQLVESGGGLVKPGGSLRLSCAASGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKFTITRDKSKNTLYLQMNSLKSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS (SEQ ID NO: 1133)Humanized 8F9A4A3 H-4.4 heavy chain variable domain sequencecaagtgcagctgcaggagagcggacctggcctggttaagcctggaggcaccctgtctctgacatgtgctgtgtctggcaatacctttaccgagtacaccatgcactgggtgcggcagcctccaggcaagggcctggaatggatcggcggcttcaaccccaacaacggcgtgacaaattacaaccagaaattcaagggaaaagtgaccatcaccgtggataagtccaagaacaccttcagcctcaagctgagcagcgtgacagccgccgacaccgccgtgtactactgcgccagaagatactatcacagcctgtacgtgttctacttcgactactggggccagggcacactggtcaccgtgtccagc (SEQ ID NO: 1105)QVQLQESGPGLVKPGGTLSLTCAVSGNTFTEYTMHWVRQPPGKGLEWIGGFNPNNGVTNYNQKFKGKVTITVDKSKNTFSLKLSSVTAADTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS (SEQ ID NO: 1106)Humanized 8F9A4A3 L-1.6 light chain variable domain sequencegatatccagatgacacagagccctagctccctgagcgccagcgtgggcgaccgggtcaccattacatgcagcgcttctcagggcatctccaactacctgaactggtaccagcagaaacccggcaaggcccctaagctgctgatcttctacaccagctctctgcacagcggcgtgccatctagattcagcggatctggcagcggcaccgactacaccctgaccatcagctccctccagcctgaggacttcgccacctactactgtcagcaatacagcaagctgccttatacctttggcggeggaacaaaggtggaaatcaag(SEQ ID NO: 1103) DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAPKLLIFYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSKLPYTFGGGTKVEIK(SEQ ID NO: 1104)Humanized 8F9A4A3 L-3.15 light chain variable domain sequencegagatcgtgatgacccagagcccagctacacttagtgtgagtccaggtgaacgggctaccctgtcctgcagcgccagccagggcatcagcaactacctgaactggtaccagcagaaacctggccaggcccctagactgctgatcttctacaccagcagcctgcacagcggcatccccgccagattcagcggcagcggctctggaacagactacaccctgacaatctctagcctgcagtctgaagattttgccgtctactactgtcagcaatacagcaagctgccttataccttcggcggcggaaccaaggtggaaattaag (SEQ ID NO: 1134) EIVMTQSPATLSVSPGERATLSCSASQGISNYLNWYQQKPGQAPRLLIFYTSSLHSGIPARFSGSGSGTDYTLTISSLQSEDFAVYYCQQYSKLPYTFGGGTKVEIK(SEQ ID NO: 1135)Humanized 8F9A4A3 L-4.1 light chain variable domain sequencegatatcgtgatgacccagagcccagacagcctggcagtgagtctgggtgagcgtgctacaatcaactgcagcgccagccagggcatctccaactacctgaattggtatcagcagaaacctggccaggctcctaagctgctgatcttctacaccagcagcctgcacagcggcgtgccagatagattcagcggcagcggatctggcaccgactacacactgaccatttettctctccaggccgaggacgtggccgtctactactgtcagcaatacagcaagctgccttacacctttggcggaggcacaaaggtggaaatcaag (SEQ ID NO: 1107)DIVMTQSPDSLAVSLGERATINCSASQGISNYLNWYQQKPGQAPKLLIFYTSSLHSGVPDRFSGSGSGTDYTLTISSLQAEDVAVYYCQQYSKLPYTFGGGTKVEIK(SEQ ID NO: 1108) Monoclonal antibody 5D9E2B11Heavy chain variable region sequence H-1, 4, 7, 8, 12gtccagctgcaacagtctggacctgatctggtgaagcctgggacttcagtgaagatatcctgtaagacttctggaaacacattcactgaatacaccatgcactgggtgaagcagagccatggaaagagccttgagtggattggaggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaagacgttactaccatagtacctacgtgttctactttgactcctggggccaaggcaccactctcacagtctectca (SEQ ID NO: 396)Translated protein, wherein the underlined sequence is the complementaritydetermining region (CDR):VQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTTLTVSS (SEQ ID NO: 397) Heavy chain variable region CDR1:NTFTEYTMH (SEQ ID NO: 398) Heavy chain variable region CDR2:GFNPNNGVTNYNQKFKG (SEQ ID NO: 399) Heavy chain variable region CDR3:RYYHSTYVFYFDS (SEQ ID NO: 400) Light chain variable region sequenceK-3, 4, 5, 6, 12gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagtgcaagtcagggcattagcaattatttaaactggtttcagcagaaaccagatggaactattaagctcctgatctattacacatcaagtttacattcaggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagtaatgtggaacctgaagatattgccacttactattgtcagcagtatagtaagcttccttacacgttcggaggggggaccaagctggagataaaacgg (SEQ ID NO: 401)Translated protein: DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR(SEQ ID NO: 402)Translated protein, wherein the underlined sequence is the complementaritydetermining region (CDR):DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR(SEQ ID NO: 403) Light chain variable region CDR 1:SASQGISNYLN (SEQ ID NO: 404) Light chain variable region CDR2:YTSSLHS (SEQ ID NO: 405) Light chain variable region CDR3:QQYSKLPYT (SEQ ID NO: 406) Monoclonal antibody 5D9E10E4Heavy chain variable region sequence H-2, 4, 7, 10, 12gtccagctgcaacagtctggacctgatctggtgaagcctgggacttcagtgaagatatcctgtaagacttctggaaacacattcactgaatacaccatgcactgggtgaagcagagccatggaaagagccttgagtggattggaggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaagacgttactaccatagtacctacgtgttctactttgactcctggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 407)Translated protein, wherein the underlined sequence is the complementaritydetermining region (CDR):VQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTTLTVSS (SEQ ID NO: 408) Heavy chain variable region CDR1:NTFTEYTMH (SEQ ID NO: 409) Heavy chain variable region CDR2:GFNPNNGVTNYNQKFKG (SEQ ID NO: 410) Heavy chain variable region CDR3:RYYHSTYVFYFDS (SEQ ID NO: 411) Light chain variable region sequenceK-2, 6, 8, 14, 15gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagtgcaagtcagggcattagcaattatttaaactggtttcagcagaaaccagatggaactattaagctcctgatctattacacatcaagtttacattcaggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagtaatgtggaacctgaagatattgccacttactattgtcagcagtatagtaagcttccttacacgttcggaggggggaccaagctggagataaaacgg (SEQ ID NO: 412)Translated protein, wherein the underlined sequence is the complementaritydetermining region (CDR):DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR(SEQ ID NO: 413) Light chain variable region CDR 1:SASQGISNYLN (SEQ ID NO: 414) Light chain variable region CDR2:YTSSLHS (SEQ ID NO: 415) Light chain variable region CDR3:QQYSKLPYT (SEQ ID NO: 416) Monoclonal antibody 5D9G2C4Heavy chain variable region sequence H-4, 9, 10, 11, 13gtccagctgcaacagtctggacctgatctggtgaagcctgggacttcagtgaagatatcctgtaagacttctggaaacacattcactgaatacaccatgcactgggtgaagcagagccatggaaagagccttgagtggattggaggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaagacgttactaccatagtacctacgtgttctactttgactcctggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 417)Translated protein, wherein the underlined sequence is the complementaritydetermining region (CDR):VQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTTLTVSS (SEQ ID NO: 418) Heavy chain variable region CDR1:NTFTEYTMH (SEQ ID NO: 419) Heavy chain variable region CDR2:GFNPNNGVTNYNQKFKG (SEQ ID NO: 420) Heavy chain variable region CDR3:RYYHSTYVFYFDS (SEQ ID NO: 421) Light chain variable region sequenceK-4, 6, 7, 8, 10gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagtgcaagtcagggcattagcaattatttaaactggtttcagcagaaaccagatggaactattaagctcctgatctattacacatcaagtttacattcaggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagtaatgtggaacctgaagatattgccacttactattgtcagcagtatagtaagcttccttacacgttcggaggggggaccaagctggagataaaacgg (SEQ ID NO: 422)Translated protein, wherein the underlined sequence is the complementaritydetermining region (CDR):DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR(SEQ ID NO: 423) Light chain variable region CDR 1:SASQGISNYLN (SEQ ID NO: 424) Light chain variable region CDR2:YTSSLHS (SEQ ID NO: 425) Light chain variable region CDR3:QQYSKLPYT (SEQ ID NO: 426) Monoclonal antibody 5F3A5D4Heavy chain variable region sequence H-2, 3 ,4, 13, 15gtccagctgcaacagtctggacctgatctggtgaagcctgggacttcagtgaagatatcctgtaagacttctggaaacacattcactgaatacaccatgcactgggtgaagcagagccatggaaagagccttgagtggattggaggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaagacgttactaccatagtacctacgtgttctactttgactcctggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 427)Translated protein, wherein the underlined sequence is the complementaritydetermining region (CDR):VQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTTLTVSS (SEQ ID NO: 428) Heavy chain variable region CDR1:NTFTEYTMH (SEQ ID NO: 429) Heavy chain variable region CDR2:GFNPNNGVTNYNQKFKG (SEQ ID NO: 430) Heavy chain variable region CDR3:RYYHSTYVFYFDS (SEQ ID NO: 431) Light chain variable region sequenceK-1, 2, 3, 4, 9gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagtgcaagtcagggcattagcaattatttaaactggtttcagcagaaaccagatggaactattaagctcctgatctattacacatcaagtttacattcaggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagtaatgtggaacctgaagatattgccacttactattgtcagcagtatagtaagcttccttacacgttcggaggggggaccaagctggagataaaacgg (SEQ ID NO: 432)Translated protein, wherein the underlined sequence is the complementaritydetermining region (CDR):DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR(SEQ ID NO: 433) Light chain variable region CDR 1:SASQGISNYLN (SEQ ID NO: 434) Light chain variable region CDR2:YTSSLHS (SEQ ID NO: 435) Light chain variable region CDR3:QQYSKLPYT (SEQ ID NO: 436) Monoclonal antibody 8F9A5A1Heavy chain variable region sequence H-3, 4, 6, 10, 11atccagttggtgcagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggagagccaacatatgttgatgacttcaagggacggtttgccttctctttggaaacctctgccaccactgcctatttgcagatcaacaacctcaaaaatgaggacacgtctacatatttctgtgcaagattgagggggatacgaccgggtcccttggcttactggggccaagggactctggtcactgtctctgca (SEQ ID NO: 437)Translated protein, wherein the underlined sequence is the complementaritydetermining region (CDR):IQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYVDDFKGRFAFSLETSATTAYLQINNLKNEDTSTYFCARLRGIRPGPLAYWGQGTLVTVSA (SEQ ID NO: 438) Heavy chain variable region CDR1:YTFTNYGMN (SEQ ID NO: 439) Heavy chain variable region CDR2:WINTYTGEPTYVDDFKG (SEQ ID NO: 440) Heavy chain variable region CDR3:LRGIRPGPLAY (SEQ ID NO: 441) Light chain variable region sequenceK-1, 2, 3, 4, 5gaaattttgctcacccagtctccagcaatcatagctgcatctcctggggagaaggtcaccatcacctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaaaccaggatcctcccccaaaatatggatttatggtatatccaacctggcttctggagttcctgctcgcttcagtggcagtgggtctgggacatctttctctttcacaatcaacagcatggaggctgaagatgttgccacttattactgtcagcaaaggagtagttacccacccacgttcggaggggggaccaagctggaaataaaacgg (SEQ ID NO: 442)Translated protein, wherein the underlined sequence is the complementaritydetermining region (CDR):EILLTQSPANIAASPGEKVTITCSASSSVSYMNWYQQKPGSSPKIWIYGISNLASGVPARFSGSGSGTSFSFTINSMEAEDVATYYCQQRSSYPPTFGGGTKLEIKR (SEQ ID NO: 443)Light chain variable region CDR 1: SASSSVSYMN (SEQ ID NO: 444)Light chain variable region CDR2: GISNLAS (SEQ ID NO: 445)Light chain variable region CDR3: QQRSSYPPT (SEQ ID NO: 446)Monoclonal antibody 8H5H5G4 Heavy chain variable region sequenceH-1, 3, 5, 6, 10gtccagctgcaacagtctggacctgatctggtgaagcctgggacttcagtgaagatatcctgtaagacttctggaaacacattcactgaatacaccatgcactgggtgaagcagagccatggaaagagccttgagtggattggaggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaagacgttactaccatagtacctacgtgttctactttgactcctggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 447)Translated protein, wherein the underlined sequence is the complementaritydetermining region (CDR):VQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTTLTVSS (SEQ ID NO: 448) Heavy chain variable region CDR1:NTFTEYTMH (SEQ ID NO: 449) Heavy chain variable region CDR2:GFNPNNGVTNYNQKFKG (SEQ ID NO: 450) Heavy chain variable region CDR3:RYYHSTYVFYFDS (SEQ ID NO: 451) Light chain variable region sequenceK-2, 5, 8, 9, 15gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagtgcaagtcagggcattagcaattatttaaactggtttcagcagaaaccagatggaactattaagctcctgatctattacacatcaagtttacattcaggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagtaatgtggaacctgaagatattgccacttactattgtcagcagtatagtaagcttccttacacgttcggaggggggaccaagctggagataaaacgg (SEQ ID NO: 452)Translated protein, wherein the underlined sequence is the complementaritydetermining region (CDR):DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR(SEQ ID NO: 453) Light chain variable region CDR 1:SASQGISNYLN (SEQ ID NO: 454) Light chain variable region CDR2:YTSSLHS (SEQ ID NO: 455) Light chain variable region CDR3:QQYSKLPYT (SEQ ID NO: 456)8F9A4P3 Heavy chain variable region sequence mouseGtccagctgcaacagtctggacctgaactggtgaagcctggggcttcagtgaagatatcctgcaagacttctggaaacacattcactgaatacaccatgcactgggtgaagcagagccatggaaagagccttgagtggattggaggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaagacggtactaccatagtctctacgtgttttactttgactactggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 335)VQLQQSGPELVKPGASVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSLYVFYFDYWGQGTTLTVSS (SEQ ID NO: 1001)IGHV1-24*01 V-REGION sequence human (closest match hu antibody sequence)Caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggtttccggatacaccctcactgaattatccatgcactgggtgcgacaggctcctggaaaagggcttgagtggatgggaggttttgatcctgaagatggtgaaacaatctacgcacagaagttccagggcagagtcaccatgaccgaggacacatctacagacacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcaaca (SEQ ID NO: 336)QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCAT (SEQ ID NO: 1002)human (closest match hu antibody sequence) IGHJ4*01 J-REGION sequencetactttgactactggggccaaggaaccctggtcaccgtctcctca (SEQ ID NO: 337)YFDYWGQGTLVTVSS (SEQ ID NO: 1003)humanized heavy chain variable seq (SEQ ID NO: 1001 + SEQ ID NO: 1002 +SEQ ID NO: 1003) humanized 8F9A4P3 Heavy chain variable region sequence(DNA)caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggtttccggaaacacattcactgaatacaccatgcactgggtgcgacaggctcctggaaaagggcttgagtggatgggaggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggcagagtcaccatgaccgaggacacatctacagacacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcaagacggtactaccatagtctctacgtgttttactttgactactggggccaaggaaccctggtcaccgtctcctca (SEQ ID NO: 338)QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWMGGFNPNNGVTNYNQKFKGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS (SEQ ID NO: 1004)humanized heavy chain variable seq (codon optimized version of 1004)humanized 8F9A4P3 Heavy chain variable region sequence (codon optimized)(DNA)caggttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggacttgaatggatgggcggcttcaaccccaacaacggcgtgaccaactacaaccagaaattcaagggccgcgtgaccatgaccgaggacacaagcacagacaccgcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcctgtacgtgttctacttcgactactggggccagggcaccctggtcacagtttcttct (SEQ ID NO: 339)QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWMGGFNPNNGVTNYNQKFKGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS (SEQ ID NO: 1005)humanized heavy chain variable seq (“modified” SEQ ID NO: 1005 sequence,where modified means certain amino acids that are thought to be critical for binding orstructure have been reverted to the mouse sequence).Modified humanized 8F9A4P3 Heavy chain variable region sequencecaggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggtttccggaaacacattcactgaatacaccatgcactgggtgcgacaggctcctggaaaagggcttgagtggattggaggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggcaaagtcaccctgaccgtggacacatctagcagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcaagacggtactaccatagtctctacgtgttttactttgactactggggccaaggaaccctggtcaccgtctcctca (SEQ ID NO: 340)QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS (SEQ ID NO: 1006)humanized heavy chain variable seq (SEQ ID NO: 1006 codon optimized)Modified humanized 8F9A4P3 Heavy chain variable region sequence (codon optimized)caggttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggactggaatggatcggcggcttcaaccccaacaacggcgtgaccaactacaaccagaaattcaagggcaaagtgaccctgaccgtggacaccagcagcagcacagcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcctgtacgtgttctacttcgactactggggccagggcaccctggtcacagtttcttct (SEQ ID NO: 341)QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSS (SEQ ID NO: 1007)8F9A4P3 Light chain variable region sequence mousegaaacaactgtgacccagtetccagcatccctgtccatggctataggagaaaaagtcaccatcagatgcataaccagcactgatattgatgatgatatgaactggtaccagcagaagccaggggaacctcctaagctccttatttcagaaggcaatactcttcgtcctggagtcccatcccgattctccagcagtggctatggtacagattttgtttttacaattgaaaacatgctctcagaagatgttgcagattactactgtttgcaaagtgataacttgcctctcacgttcggctcggggacaaagttggaaataaaacgg (SEQ ID NO: 342)ETTVTQSPASLSMAIGEKVTIRCITSTDIDDDMNWYQQKPGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPLTFGSGTKLEIKR(SEQ ID NO: 1008) human (closest match hu antibody sequence)IGKV5-2*01 V-REGION sequencegaaacgacactcacgcagtetccagcattcatgtcagcgactccaggagacaaagtcaacatctcctgcaaagccagccaagacattgatgatgatatgaactggtaccaacagaaaccaggagaagctgctattttcattattcaagaagctactactctcgttcctggaatcccacctcgattcagtggcagcgggtatggaacagattttaccctcacaattaataacatagaatctgaggatgctgcatattacttctgt (SEQ ID NO: 343)ETTLTQSPAFMSATPGDKVNISCKASQDIDDDMNWYQQKPGEAAIFIIQEATTLVPGIPPRFSGSGYGTDFTLTINNIESEDAAYYFC (SEQ ID NO: 1009)human (closest match hu antibody sequence) IGKJ4*02 J-REGION sequencectcacgttcggcggagggaccaaggtggagatcaaa (SEQ ID NO: 344)LTFGGGTKVEIK (SEQ ID NO: 1010)humanized light chain variable seq (SEQ ID NO: 1008 + SEQ ID NO: 1009 + SEQ ID NO: 1)humanized 8F9A4P3 Light chain variable region sequencegaaacgacactcacgcagtetccagcattcatgtcagcgactccaggagacaaagtcaacatctcctgcataaccagcactgatattgatgatgatatgaactggtaccaacagaaaccaggagaagctgctattttcattattcaagaaggcaatactcttcgtcctggaatcccacctcgattcagtggcagcgggtatggaacagattttaccctcacaattaataacatagaatctgaggatgctgcatattacttctgtttgcaaagtgataacttgcctctcacgttcggcggagggaccaaggtggagatcaaacgg (SEQ ID NO: 345)ETTLTQSPAFMSATPGDKVNISCITSTDIDDDMNWYQQKPGEAAIFIIQEGNTLRPGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCLQSDNLPLTFGGGTKVEIKR (SEQID NO: 1011)humanized light chain variable seq (codon optimized version of SEQ ID NO: 1011)humanized 8F9A4P3 Light chain variable region sequence (codon optimized)Gagacaaccctgacacagagccctgccttcatgtctgccacacctggcgacaaagtgaacatcagctgcatcaccagcaccgacatcgacgacgacatgaactggtatcagcagaagcctggcgaggccgccatcttcatcatccaagagggcaacacactgcggcctggcatccctcctagattttctggcagcggctacggcaccgacttcaccctgaccatcaacaacatcgagagcgaggacgccgcctactacttctgcctgcaaagcgacaacctgcctctgacctttggcggaggcaccaaggtggaaatcaagcgg (SEQ IDNO: 346) ETTLTQSPAFMSATPGDKVNISCITSTDIDDDMNWYQQKPGEAAIFIIQEGNTLRPGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCLQSDNLPLTFGGGTKVEIKR (SEQID NO: 1012)humanized light chain variable seq (“modified” SEQ ID NO: 1012 sequence,where modified means certain amino acids that are thought to be critical for binding orstructure have been reverted to the mouse sequence).Modified humanized 8F9A4P3 Light chain variable region sequencegaaacgacagtgacgcagtetccagcattcatgtcagcgactccaggagacaaagtcaccatctcctgcataaccagcactgatattgatgatgatatgaactggtaccaacagaaaccaggagaagctgctattctgctgattagcgaaggcaatactcttcgtcctggaatcccacctcgattcagtagcagcgggtatggaacagattttaccctcacaattaataacatagaatctgaggatgctgcatattacttctgtttgcaaagtgataacttgcctctcacgttcggcggagggaccaaggtggagatcaaacgg (SEQ ID NO: 347)ETTVTQSPAFMSATPGDKVTISCITSTDIDDDMNWYQQKPGEAAILLISEGNTLRPGIPPRFSSSGYGTDFTLTINNIESEDAAYYFCLQSDNLPLTFGGGTKVEIKR(SEQ ID NO: 1013)humanized light chain variable seq (SEQ ID NO: 1013 codon optimized)Modified humanized 8F9A4P3 Light chain variable region sequence (codon optimized)gagacaaccgtgacacagagccctgccttcatgtctgccacacctggcgacaaagtgaccatcagctgcatcaccagcaccgacatcgacgacgacatgaactggtatcagcagaagcctggcgaggccgccatcctgcttatctctgagggaaacacactgcggcctggcatccctcctagattttccagcagcggctacggcaccgacttcaccctgaccatcaacaacatcgagagcgaggacgccgcctactacttctgcctgcaaagcgacaacctgcctctgacctttggcggaggcaccaaggtggaaatcaagcgg(SEQ ID NO: 348) ETTVTQSPAFMSATPGDKVTISCITSTDIDDDMNWYQQKPGEAAILLISEGNTLRPGIPPRFSSSGYGTDFTLTINNIESEDAAYYFCLQSDNLPLTFGGGTKVEIKR(SEQ ID NO: 1014)humanized heavy and light chains joined via a flexible linker.Modified humanized 8F9A4P3 sequence (codon optimized)Caggttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggactggaatggatcggcggcttcaaccccaacaacggcgtgaccaactacaaccagaaattcaagggcaaagtgaccctgaccgtggacaccagcagcagcacagcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcctgtacgtgttctacttcgactactggggccagggcaccctggtcacagtttcttctggcggtggcggaagcggaggcggtggctccggtggcggaggcagcgaaacgacagtgacgcagtctccagcattcatgtcagcgactccaggagacaaagtcaccatctcctgcataaccagcactgatattgatgatgatatgaactggtaccaacagaaaccaggagaagctgctattctgctgattagcgaaggcaatactcttcgtcctggaatcccacctcgattcagtagcagcgggtatggaacagattttaccctcacaattaataacatagaatctgaggatgctgcatattacttctgtttgcaaagtgataacttgcctctcacgttcggcggagggaccaaggtggagatcaaacgg (SEQ ID NO: 349)QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSLYVFYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSETTVTQSPAFMSATPGDKVTISCITSTDIDDDMNWYQQKPGEAAILLISEGNTLRPGIPPRFSSSGYGTDFTLTINNIESEDAAYYFCLQSDNLPLTFGGGTKVEIKR (SEQ ID NO: 1015)8F9A5A1 Heavy chain variable region sequenceatccagttggtgcagtctggacctgagctgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggagagccaacatatgttgatgacttcaagggacggtttgccttctctttggaaacctctgccaccactgcctatttgcagatcaacaacctcaaaaatgaggacacgtctacatatttctgtgcaagattgagggggatacgaccgggtcccttggcttactggggccaagggactctggtcactgtctctgca (SEQ ID NO: 350)IQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYVDDFKGRFAFSLETSATTAYLQINNLKNEDTSTYFCARLRGIRPGPLAYWGQGTLVTVSA (SEQ ID NO: 1016) IGHV7-81*01 V-REGION sequencecaggtgcagctggtgcagtctggccatgaggtgaagcagcctggggcctcagtgaaggtctcctgcaaggcttctggttacagtttcaccacctatggtatgaattgggtgccacaggcccctggacaagggcttgagtggatgggatggttcaacacctacactgggaacccaacatatgcccagggcttcacaggacggtttgtcttctccatggacacctctgccagcacagcatacctgcagatcagcagcctaaaggctgaggacatggccatgtattactgtgcgaga (SEQ ID NO: 351)QVQLVQSGHEVKQPGASVKVSCKASGYSFTTYGMNWVPQAPGQGLEWMGWFNTYTGNPTYAQGFTGRFVFSMDTSASTAYLQISSLKAEDMAMYYCAR (SEQ ID NO: 1017)IGHJ4*03 J-REGION sequencetactttgactactggggccaagggaccctggtcaccgtctcctca (SEQ ID NO: 352)YFDYWGQGTLVTVSS (SEQ ID NO: 1018)humanized 8F9A5A1 Heavy chain variable region sequenceCaggtgcagctggtgcagtctggccatgaggtgaagcagcctggggcctcagtgaaggtctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgccacaggcccctggacaagggcttgagtggatgggatggataaacacctacactggagagccaacatatgttgatgacttcaagggacggtttgtcttctccatggacacctctgccagcacagcatacctgcagatcagcagcctaaaggctgaggacatggccatgtattactgtgcaagattgagggggatacgaccgggtcccttggcttactggggccaagggaccctggtcaccgtctcctca (SEQ ID NO: 353)QVQLVQSGHEVKQPGASVKVSCKASGYTFTNYGMNWVPQAPGQGLEWMGWINTYTGEPTYVDDFKGRFVFSMDTSASTAYLQISSLKAEDMAMYYCARLRGIRPGPLAYWGQGTLVTVSS (SEQ ID NO: 1019)humanized 8F9A5A1 Heavy chain variable region sequence (codon optimized)caggttcagctggtgcagtctggccacgaagtgaaacagcctggcgcctctgtgaaggtgtcctgtaaagccagcggctacacctttaccaactacggcatgaactgggtgccccaggctcctggacaaggcttggaatggatgggctggatcaacacctacaccggcgagcctacctacgtggacgacttcaagggcagattcgtgttcagcatggacaccagcgccagcacagcctacctgcagatcagctctctgaaggccgaggatatggccatgtactactgcgccagactgagaggcatcagacctggacctctggcctattggggacagggcacactggtcacagtgtcctct (SEQ ID NO: 354)QVQLVQSGHEVKQPGASVKVSCKASGYTFTNYGMNWVPQAPGQGLEWMGWINTYTGEPTYVDDFKGRFVFSMDTSASTAYLQISSLKAEDMAMYYCARLRGIRPGPLAYWGQGTLVTVSS (SEQ ID NO: 1020)Modified humanized 8F9A5A1 Heavy chain variable region sequencecagatccagctggtgcagtctggccccgaggtgaagcagcctggggcctcagtgaaggtctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggcccctggacaagggcttgagtggatgggatggataaacacctacactggagagccaacatatgttgatgacttcaagggacggtttgccttctccatggacacctctgccagcacagcatacctgcagatcagcagcctaaaggctgaggacaccgccacctattactgtgcaagattgagggggatacgaccgggtcccttggcttactggggccaagggaccctggtcaccgtctcctca (SEQ ID NO: 355)QIQLVQSGPEVKQPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGEPTYVDDFKGRFAFSMDTSASTAYLQISSLKAEDTATYYCARLRGIRPGPLAYWGQGTLVTVSS (SEQ ID NO: 1021)Modified humanized 8F9A5A1 Heavy chain variable region sequence (codon optimized)cagattcagctggtgcagtctggccccgaagtgaaacaacctggcgcctctgtgaaggtgtcctgcaaggccagcggctacacctttaccaactacggcatgaactgggtcaagcaggcccctggacaaggcctggaatggatgggctggatcaacacctacaccggcgagcctacctacgtggacgacttcaagggcagattcgccttcagcatggacaccagcgccagcacagcctacctgcagatcagctctctgaaggccgaggacaccgccacctactactgtgccagactgagaggcatcagacccggacctctggcctattggggacagggaacactggtcaccgtgtcctct (SEQ ID NO: 356)QIQLVQSGPEVKQPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGEPTYVDDFKGRFAFSMDTSASTAYLQISSLKAEDTATYYCARLRGIRPGPLAYWGQGTLVTVSS (SEQ ID NO: 1022)8F9A5A1 Light chain variable region sequencegaaattttgctcacccagtctccagcaatcatagctgcatctcctggggagaaggtcaccatcacctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaaaccaggatcctcccccaaaatatggatttatggtatatccaacctggcttctggagttcctgctcgcttcagtggcagtgggtctgggacatctttctctttcacaatcaacagcatggaggctgaagatgttgccacttattactgtcagcaaaggagtagttacccacccacgttcggaggggggaccaagctggaaataaaacgg (SEQ ID NO: 357)EILLTQSPANIAASPGEKVTITCSASSSVSYMNWYQQKPGSSPKIWIYGISNLASGVPARFSGSGSGTSFSFTINSMEAEDVATYYCQQRSSYPPTFGGGTKLEIKR (SEQ ID NO: 1023)IGKV3D-15*02 V-REGION sequencegaaatagtgatgatgcagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagcaacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatggtgcatccaccagggccactggcatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgtcagcagtataataac (SEQ ID NO: 358)EIVMMQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNN (SEQ ID NO: 1024)IGKJ4*02 J-REGION sequencectcacgttcggcggagggaccaaggtggagatcaaa (SEQ ID NO: 359)LTFGGGTKVEIK (SEQ ID NO: 1025)humanized 8F9A5A1 Light chain variable region sequencegaaatagtgatgatgcagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaaacctggccaggctcccaggctcctcatctatggtatatccaacctggcttctggcatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgtcagcaaaggagtagttacccacccacgttcggcggagggaccaaggtggagatcaaacgg (SEQ ID NO: 360)EIVMMQSPATLSVSPGERATLSCSASSSVSYMNWYQQKPGQAPRLLIYGISNLASGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQRSSYPPTFGGGTKVEIKR (SEQID NO: 1026)humanized 8F9A5A1 Light chain variable region sequence (codon optimized)gagatcgtgatgatgcagagccccgccacactgagtgtgtctccaggcgaaagagccacactgtcctgtagcgccagcagcagcgtgtcctacatgaactggtatcagcagaagcccggacaggcccctagactgctgatctacggcatcagcaatctggccagcggcatccctgccagattttctggctctggctccggcaccgagttcaccctgacaatctctagcctgcagagcgaggacttcgccgtgtactactgccagcagagaagcagctaccctcctacctttggcggaggcaccaaggtggaaatcaagcgg (SEQ ID NO: 361)EIVMMQSPATLSVSPGERATLSCSASSSVSYMNWYQQKPGQAPRLLIYGISNLASGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQRSSYPPTFGGGTKVEIKR (SEQID NO: 1027)Modified humanized 8F9A5A1 Light chain variable region sequencegaaatagtgctgacccagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagtgccagctcaagtgtaagttacatgaactggtaccagcagaaacctggccaggctcccaggctctggatctatggtatatccaacctggcttctggcatcccagccaggttcagtggcagtgggtctgggacaagcttcagcctcaccatcagcagcctgcagtctgaagattttgcagtttattactgtcagcaaaggagtagttacccacccacgttcggcggagggaccaaggtggagatcaaacgg (SEQ ID NO: 362)EIVLTQSPATLSVSPGERATLSCSASSSVSYMNWYQQKPGQAPRLWIYGISNLASGIPARFSGSGSGTSFSLTISSLQSEDFAVYYCQQRSSYPPTFGGGTKVEIKR (SEQID NO: 1028)Modified humanized 8F9A5A1 Light chain variable region sequence (codon optimized)gagatcgtgctgacacagtctcccgccacactgagtgtgtctccaggcgaaagagccacactgtcctgtagcgccagcagcagcgtgtcctacatgaactggtatcagcagaagcccggacaggcccctagactgtggatctacggcatcagcaatctggccagcggcatccctgccagattttctggctctggctccggcaccagcttcagcctgacaatcagcagcctgcagagcgaggacttcgccgtgtactactgccagcagagaagcagctaccctcctacctttggcggaggcaccaaggtggaaatcaagcgg (SEQ ID NO: 363)(amino acids) EIVLTQSPATLSVSPGERATLSCSASSSVSYMNWYQQKPGQAPRLWIYGISNLASGIPARFSGSGSGTSFSLTISSLQSEDFAVYYCQQRSSYPPTFGGGTKVEIKR (SEQID NO: 1029) Modified humanized 8F9A5A1 scFV sequence (codon optimized)Cagattcagctggtgcagtctggccccgaagtgaaacaacctggcgcctctgtgaaggtgtcctgcaaggccagcggctacacctttaccaactacggcatgaactgggtcaagcaggcccctggacaaggcctggaatggatgggctggatcaacacctacaccggcgagcctacctacgtggacgacttcaagggcagattcgccttcagcatggacaccagcgccagcacagcctacctgcagatcagctctctgaaggccgaggacaccgccacctactactgtgccagactgagaggcatcagacccggacctctggcctattggggacagggaacactggtcaccgtgtcctctggcggtggcggaagcggaggcggtggctccggtggcggaggcagcgagatcgtgctgacacagtctcccgccacactgagtgtgtctccaggcgaaagagccacactgtcctgtagcgccagcagcagcgtgtcctacatgaactggtatcagcagaagcccggacaggcccctagactgtggatctacggcatcagcaatctggccagcggcatccctgccagattttctggctctggctccggcaccagcttcagcctgacaatcagcagcctgcagagcgaggacttcgccgtgtactactgccagcagagaagcagctaccctcctacctttggcggaggcaccaaggtggaaatcaagcgg (SEQ ID NO: 364)QIQLVQSGPEVKQPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGEPTYVDDFKGRFAFSMDTSASTAYLQISSLKAEDTATYYCARLRGIRPGPLAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSVSPGERATLSCSASSSVSYMNWYQQKPGQAPRLWIYGISNLASGIPARFSGSGSGTSFSLTISSLQSEDFAVYYCQQRSSYPPTFGGGTKVEIKR (SEQ ID NO: 1030)8H5H5G4 Heavy chain variable region sequencegtccagctgcaacagtctggacctgatctggtgaagcctgggacttcagtgaagatatcctgtaagacttctggaaacacattcactgaatacaccatgcactgggtgaagcagagccatggaaagagccttgagtggattggaggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccagcacagcctacatggagctccgcagcctgacatctgaggattctgcagtctattactgtgcaagacgttactaccatagtacctacgtgttctactttgactcctggggccaaggcaccactctcacagtctcctca (SEQ ID NO: 365)VQLQQSGPDLVKPGTSVKISCKTSGNTFTEYTMHWVKQSHGKSLEWIGGFNPNNGVTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARRYYHSTYVFYFDSWGQGTTLTVSS (SEQ ID NO: 1031) IGHV1-24*01 V-REGION sequence (DNA)caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggtttccggatacaccctcactgaattatccatgcactgggtgcgacaggctcctggaaaagggcttgagtggatgggaggttttgatcctgaagatggtgaaacaatctacgcacagaagttccagggcagagtcaccatgaccgaggacacatctacagacacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcaaca (SEQ ID NO: 366)(amino acids) QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCAT (SEQ ID NO: 1032)IGHJ4*03 J-REGION sequence (DNA)tactttgactactggggccaagggaccctggtcaccgtctcctca (SEQ ID NO: 367)(amino acids) YFDYWGQGTLVTVSS (SEQ ID NO: 1033)Humanized 8H5H5G4 Heavy chain variable region sequence (DNA)caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggtttccggaaacacattcactgaatacaccatgcacTgggtgcgacaggctcctggaaaagggcttgagtggatgggaggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggcAgagtcaccatgaccgaggacacatctacagacacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtGcaagacgttactaccatagtacctacgtgttctactttgactcctggggccaagggaccctggtcaccgtctcctca (SEQ ID NO: 368) (amino acids)QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWMGGFNPNNGVTNYNQKFKGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSS (SEQ ID NO: 1034)Humanized 8H5H5G4 Heavy chain variable region sequence (codon optimized)(DNA)caggttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggacttgaatggatgggcggcttcaaccccaacaacggcgtgaccaactacaaccagaaattcaagggccgcgtgaccatgaccgaggacacaagcacagacaccgcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcacctacgtgttctacttcgacagctggggccagggcacactggtcacagtttcttct (SEQ ID NO: 369) (amino acids)QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWMGGFNPNNGVTNYNQKFKGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSS (SEQ ID NO: 1035)Modified Humanized 8H5H5G4 Heavy chain variable region sequence (DNA)caggtccagctggtacagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggtttccggaaacacattcactgaatacaccatgcactgggtgcgacaggctcctggaaaagggcttgagtggatcggaggttttaatcctaacaatggtgttactaactacaaccagaagttcaagggcaaggtcaccctgaccgtggacacatctagcagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcaagacgttactaccatagtacctacgtgttctactttgactcctggggccaagggaccctggtcaccgtctcctca (SEQ ID NO: 370) (amino acids)QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSS (SEQ ID NO: 1036)Modified Humanized 8H5H5G4 Heavy chain variable region sequence (codon optimized)(DNA)caggttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggactggaatggatcggcggcttcaaccccaacaacggcgtgaccaactacaaccagaaattcaagggcaaagtgaccctgaccgtggacaccagcagcagcacagcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcacctacgtgttctacttcgacagctggggccagggcacactggtcacagtttcttct (SEQ ID NO: 371) (amino acids)QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSS (SEQ ID NO: 1037)8H5H5G4 Light chain variable region sequence (DNA)gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagtgcaagtcagggcattagcaattatttaaactggtttcagcagaaaccagatggaactattaagctcctgatctattacacatcaagtttacattcaggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagtaatgtggaacctgaagatattgccacttactattgtcagcagtatagtaagcttccttacacgttcggaggggggaccaagctggagataaaacgg (SEQ ID NO: 372)(amino acids) DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTIKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNVEPEDIATYYCQQYSKLPYTFGGGTKLEIKR(SEQ ID NO: 1038) IGKV1-27*01 V-REGION sequence (DNA)gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcgagtcagggcattagcaattatttagcctggtatcagcagaaaccagggaaagttcctaagctcctgatctatgctgcatccactttgcaatcaggggtcccatctcggttcagtggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtcaaaagtataacagtgcccct (SEQ ID NO: 373) (amino acids)DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAP (SEQ ID NO: 1039)IGKJ4*02 J-REGION sequence (DNA)ctcacgttcggcggagggaccaaggtggagatcaaa (SEQ ID NO: 374) (amino acids)LTFGGGTKVEIK (SEQ ID NO: 1040)humanized 8H5H5G4 Light chain variable region sequence (DNA)gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcagtgcaagtcagggcattagcaattatttaaacTggtatcagcagaaaccagggaaagttcctaagctcctgatctattacacatcaagtttacattcaggggtcccatctcggttcagtggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtcagcagtatagtaagcttccttacacgttcggcggagggaccaaggtggagatcaaacgg (SEQ ID NO: 375)(amino acids) DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKVPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSKLPYTFGGGTKVEIKR(SEQ ID NO: 1041)humanized 8H5H5G4 Light chain variable region sequence (codon optimized)(DNA)gacatccagatgacacagagccctagcagcctgtctgccagcgtgggagacagagtgaccatcacatgtagcgccagccagggcatcagcaactacctgaactggtatcagcagaaacccggcaaggtgcccaagctgctgatctactacaccagcagcctgcacagcggcgtgccaagcagattttctggcagcggctctggcaccgacttcaccctgaccatatctagcctgcagcctgaggacgtggccacctactactgtcagcagtacagcaagctgccctacacctttggcggaggcaccaaggtggaaatcaagcgg (SEQ IDNO: 376) (amino acids)DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKVPKLLIYYTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSKLPYTFGGGTKVEIKR(SEQ ID NO: 1042)Modified humanized 8H5H5G4 Light chain variable region sequence (DNA)gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgcagtgcaagtcagggcattagcaattatttaaactggtatcagcagaaaccagggaaagttcctaagctcctgatctattacacatcaagtttacattcaggggtcccatctcggttcagtggcagtggatctgggacagattacactctcaccatcagcagcctgcagcctgaagatgttgcaacttattactgtcagcagtatagtaagcttccttacacgttcggcggagggaccaaggtggagatcaaacgg (SEQ ID NO: 377)(amino acids) DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKVPKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQYSKLPYTFGGGTKVEIKR(SEQ ID NO: 1043)Modified humanized 8H5H5G4 Light chain variable region sequence (codon optimized)(DNA)gacatccagatgacacagagccctagcagcctgtctgccagcgtgggagacagagtgaccatcacatgtagcgccagccagggcatcagcaactacctgaactggtatcagcagaaacccggcaaggtgcccaagctgctgatctactacaccagcagcctgcacagcggcgtgccaagcagattttctggcagcggctctggcaccgactacaccctgaccatatctagcctgcagcctgaggacgtggccacctactactgtcagcagtacagcaagctgccctacacctttggcggaggcaccaaggtggaaatcaagcgg (SEQ IDNO: 378) (amino acids)DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKVPKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQYSKLPYTFGGGTKVEIKR(SEQ ID NO: 1044)Modified humanized 8H5H5G4 scFV sequence (codon optimized) (DNA)Caggttcagctggttcagtctggcgccgaagtgaagaaacctggcgcctctgtgaaggtgtcctgcaaggtgtccggaaataccttcaccgagtacaccatgcactgggtccgacaggcccctggcaaaggactggaatggatcggcggcttcaaccccaacaacggcgtgaccaactacaaccagaaattcaagggcaaagtgaccctgaccgtggacaccagcagcagcacagcctacatggaactgagcagcctgagaagcgaggacaccgccgtgtactactgcgccagaaggtactaccacagcacctacgtgttctacttcgacagctggggccagggcacactggtcacagtttcttctggcggtggcggaagcggaggcggtggctccggtggcggaggcagcgacatccagatgacacagagccctagcagcctgtctgccagcgtgggagacagagtgaccatcacatgtagcgccagccagggcatcagcaactacctgaactggtatcagcagaaacccggcaaggtgcccaagctgctgatctactacaccagcagcctgcacagcggcgtgccaagcagattttctggcagcggctctggcaccgactacaccctgaccatatctagcctgcagcctgaggacgtggccacctactactgtcagcagtacagcaagctgccctacacctttggcggaggcaccaaggtggaaatcaagcgg (SEQ ID NO: 379)(amino acids) QVQLVQSGAEVKKPGASVKVSCKVSGNTFTEYTMHWVRQAPGKGLEWIGGFNPNNGVTNYNQKFKGKVTLTVDTSSSTAYMELSSLRSEDTAVYYCARRYYHSTYVFYFDSWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKVPKLLIYYTSSLHSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQYSKLPYTFGGGTKVEIKR (SEQ ID NO: 1045)Human IgG1 heavy chain constant region sequence: (for making full antibody-pair with either kappa or lambda constant region; 2 plasmids, express together)(DNA)gctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggetgtcctacagtcctcaggactctactccctcagcagcgtggtgacagtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 380)(amino acids) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1046)Human IgG2 heavy chain constant region sequence: (for making full antibody-pair with either kappa or lambda constant region; 2 plasmids, express together)(DNA)gcctccaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagccgccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagectgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatag (SEQ ID NO: 381)(amino acids) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1047)Human Kappa light chain constant region sequence: (DNA)aggacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag(SEQ ID NO: 382) (amino acids)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 1048) Human Lambda light chain constant region sequence:(DNA)ggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttcatag(SEQ ID NO: 383) (amino acids)GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO: 1049)Human IgG1 Fc region sequence: (to be fused to scFv for homo-dimerizes)(DNA)gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga (SEQ ID NO: 384) (amino acids)EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* (SEQ ID NO: 1050) Human IgG2 Fc region sequence: (DNA)gagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatag (SEQ ID NO: 385) (amino acids)ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* (SEQ ID NO: 1051)

In another aspect of the invention, an immune cell engineered to expressa CAR is administered to a patient diagnosed with or at risk ofdeveloping a cancer or cancer metastasis, wherein the immune cell isalso engineered to express an anti-NME7 antibody or antibody fragment,which may be expressed off of an inducible promoter. In one aspect, theCAR is guided by an anti-MUC1* antibody fragment. In one case, the CARis huMNC2-CAR44. In one aspect, the anti-NME7 antibody or antibodyfragment binds to an NME peptide listed under sections “Homologouspeptides to A1, A2, B1, B2 or B3 peptides” and the “Homologous extendedpeptides to A1, A2, B1, B2 or B3 peptides” above. In another aspect, theantibody or antibody fragment binds to an NME7 derived peptide chosenfrom among A1, A2, B1, B2 or B3 (SEQ ID NOS: 141-145). In yet anotheraspect, the antibody, antibody fragment or antibody mimic binds to anNME7 peptide comprising the B3 peptide. In one aspect of the invention,the anti-NME7 antibody, antibody fragment or antibody mimic comprisessequences derived from the variable domains of anti-NME7 antibodies8F9A4A3, 8F9A5A1, or 8H5H5G4.

Such antibodies may be human or humanized. Such antibodies may bepolyclonal, monoclonal, bispecific, bivalent, monovalent, single chain,scFv, or may be an antibody mimic such as protein scaffolds that presentrecognition regions that bind to a specific target. As is appreciated bythose skilled in the art, antibodies can be of non-human origin, humanor humanized. Methods for humanizing antibodies include fusing all orsome of the mouse variable regions to V- and J-regions of a closestmatch human antibody sequence, for example, as shown in sequences listedas SEQ ID NOS:1001-1045. Full antibodies, rather than single chainconstructs, can also be made. For example, the heavy chain variablemouse sequence is fused to human V- and J-regions then fused to thehuman heavy chain constant regions of IgG1, IgG2 or IgG3. Similarly, thelight chain variable mouse sequences are fused to human V- and J-regionsthen fused to either the human Kappa or Lambda constant regions of IgG1,IgG2 or IgG3. Plasmids are expressed together and associate to form thefull antibody (SEQ ID NOS:1047-1051).

In another aspect of the invention, small molecules are anti-canceragents that are selected for their ability to inhibit the tumorigeniceffects of NME7, NME7_(AB) or NME7-X1. For example, a high throughputscreen identifies small molecules that will treat cancer. In amulti-well plate, small molecules are separately added to wells in whichcancer cells are cultured in a medium containing NME7_(AB). If the smallmolecule diminishes the amount of cells that become floaters and/orreduces the expression of metastatic markers such as CXCR4, CHD1 orpluripotent stem cell markers, then that small molecule is ananti-cancer drug candidate. Another method of identifying smallmolecules that are anti-cancer agents is to select those small moleculesthat bind to NME7, NME7_(AB) or NME7-X1 or suppresses expression of theNME7 species. Yet another high throughput screen is to select for smallmolecules that inhibit the binding of NME7_(AB) to the PSMGFR peptide ofthe MUC1* extracellular domain and those small molecules will beanti-cancer agents.

The sequences of NME7_(AB) and NME7-X1 differ only in that NME7-X1 ismissing some of the N-terminal sequence that NME7_(AB) has. Experimentsshow that there is a naturally occurring NME7 species that is nearlyidentical to NME7_(AB), which we call NME7_(AB)-like species. Antibodiesthat bind to NME7-X1 may also bind to the naturally occurring speciesthat mimics NME7_(AB), unless there are conformational differences thatan antibody can differentiate. Therefore, if it is desired to inhibitNME7-X1 but not NME7_(AB)-like species, or vice versa, siRNA, anti-sensenucleic acids, or genetic editing techniques can be used to inhibitexpression of one but not the other.

In one case, the anti-cancer therapeutic agent is a nucleic acid thatdirectly or indirectly suppresses specific expression of NME7, NME7-X1or NME7_(AB)-like species. Such nucleic acids can be siRNA, RNAi,anti-sense nucleic acids and the like that directly suppress the NME7species. In another aspect of the invention, the nucleic acid canindirectly suppress the NME7 species for example by altering theexpression of a molecule that regulates it. For example, the superenhancer BRD4 suppresses expression of NME7. Therefore, an effectivetherapeutic for the treatment or prevention of cancer is an agent thatincreases expression of BRD4. An effective therapeutic may be an agentthat increases expression of BRD4's co-factor, JMJD6.

Peptides derived from NME7_(AB) or NME7-X1, or the entire protein, areused to generate anti-NME7 or anti-NME7-X1 antibodies in animals that wehave demonstrated inhibit cancer growth and inhibit transition of cancercells to metastatic cancer cells. Similarly, NME7 derived peptides canbe administered to a human such that they generate antibodies that treator prevent cancer or inhibit transition of cancer cells to metastaticcancer cells. NME7 peptides or proteins are administered to a person asa type of vaccine to stimulate the production of anti-NME7,anti-NME7_(AB) or anti-NME7-X1 antibodies in the recipient. The resultsshown in FIG. 12 and FIG. 13 indicate that immunizing a person with acollection of peptides derived from NME7, especially in the NME7-X1 orNME7_(AB) sequences may be a more effective vaccine than immunizing witha single peptide. Said peptides or proteins may further be conjugated toa carrier protein or other adjuvant, known to those skilled in the artto aid in the stimulation of an immune response.

NME7 peptides that lie outside of the DM10 domain are preferred togenerate antibodies for the treatment or prevention of cancer. Peptidesthat can be administered to a patient for the prevention of cancer ormetastasis contain sequences of the peptides listed in FIG. 6 -FIG. 9 .A1, A2, B1, B2 and B3 are examples of peptides that generate antibodiesthat bind to NME7_(AB) and NME7-X1 and are administered to a patient forthe treatment or prevention of cancer. The invention is not limited topeptides of the exact sequence as is naturally occurring in NME7 orNME7-X1. As is known to those skilled in the art, substitution ofseveral amino acids of a peptide sequence can still give rise toantibodies that specifically recognize the natural protein sequence. Itis not intended that the invention be limited to the peptidesdemonstrated herein to inhibit cancer growth or inhibit the transitionof regular cancer cells to metastatic cancer cells. The methods usedhere to identify peptides A1, A2, B1, B2 and B3 can also be used toidentify other peptide sequences that could be equally or more effectivethan the peptides demonstrated here.

Chimeric antigen receptor molecules comprising portions of humanNME7_(AB) or NME7-X1 or comprising an antibody fragment that binds toNME7_(AB) or NME7-X1 are anti-cancer therapeutics and are administeredto a patient for the treatment or prevention of cancers or cancermetastases.

In one instance, the recognition units or variable regions of anti-NME7antibodies are fused to molecules of T cells using the technology knownas CAR (chimeric antigen receptor) technology or CAR T technology. Thesalient feature of antibodies or fragments thereof that can be usedtherapeutically to treat or prevent cancers is the identification ofantibody-like variable regions that recognize NME7 and prevent itsinteraction with targets that promote cancers. In one case, the targetis the PSMGFR region of MUC1*.

Antibodies, antibody fragments or single chain antibodies can beengineered into chimeric molecules, including chimeric antigenreceptors, also known as CARs, which molecules are then transfected ortransduced into an immune system cell, such as a T cell, andadministered to a patient. The humanized antibodies or antibodyfragments, typically an scFv, comprises much of the extracellular domainof a CAR. The antibody fragment is biochemically fused to immune systemsignaling molecules, such as CD8 as the transmembrane domain andcytoplasmic signaling motifs such as T cell receptor signaling moleculesalso called activation domains, or co-stimulatory domains including butnot limited to CD3-zeta, CD28, 41bb, OX40. CARs can be transfected intoT cells or other cells, preferably immune system cells and administeredto a patient. Here we describe CARs in which the extracellular portioncontains an anti-NME7, anti-NME7_(AB) or anti-NME7-X1 antibody, antibodyfragment or single chain, scFv antibody fragment. In a preferredembodiment, the antibody or antibody fragment is human or humanized.

Effective anti-NME7 or anti-NME7-X1 antibodies or fragments will havethe ability to bind to native NME7, NME7_(AB) or NME7-X1. In practice,the parent antibody, from which the extracellular domain of the CAR isengineered, is generated by immunizing an animal with an NME7, NME7_(AB)or NME7-X1 derived peptide. In one aspect of the invention, theimmunizing peptide is comprised of NME7 amino acids 1-376. In one aspectof the invention, the immunizing peptide is comprised of NME7 aminoacids 92-376. In another aspect of the invention, the immunizing peptideis comprised of NME7 amino acids 125-376. In yet another aspect of theinvention, the immunizing peptide is made up of sequences listed in FIG.6 -FIG. 8 . In another aspect of the invention, the immunizing peptideis made up of sequences listed in FIG. 9 . Alternatively, the parentantibody or the antibody fragment is selected from a library or pool ofantibodies, which may be natural, synthetic or fragments of either,wherein they are selected for their ability to bind to NME7, NME7_(AB)or NME7-X1, peptides listed in FIG. 6 -FIG. 8 , or peptides listed inFIG. 9 .

The targeting portion of a CAR need not be an antibody or antibodyfragment. Here we describe a CAR wherein the extracellular domaincontains an NME7 fragment. NME7-derived peptide(s) are engineered into adifferent sort of CAR wherein the targeting portion of the extracellulardomain is a protein fragment or peptide rather than an antibody orantibody fragment. The peptide CARs are transfected or transduced intoan immune system cell, typically a T cell. The NME7 fragments or NME7derived peptides are selected for their ability to bind to their cognatebinding partners but should not be able to function as intact NME7,NME7_(AB) or NME7-X1 and confer tumorigenic activity. NME7 fragments orNME7 derived peptides are biochemically fused to immune system signalingmolecules, such as CD8 as the transmembrane domain and cytoplasmicsignaling motifs such as T cell receptor signaling molecules also calledactivation domains, or co-stimulatory domains including but not limitedto CD3-zeta, CD28, 41bb, OX40.

In one aspect of the invention, the NME7 fragment is most or all of theNME7 NDPK B domain. In another aspect of the invention, the NME7fragment is an NME7 peptide that contains one or more of the peptidesequences listed in FIG. 6 -FIG. 9 . Experiments indicate that, forstrategies that use NME7 or fragments of NME7, NME7_(AB), or NME7-X1 asthe targeting portion of a chimeric antigen receptor (CAR) forengineered immune cell therapeutics, fairly large fragments of NME7_(AB)or NME7-X1 would be more effective than shorter peptides, for examplepeptides less than 15 amino acids in length. Alternatively, a collectionof CARs, each bearing a different NME7_(AB) derived peptide cancollectively be transfected or transduced into an immune system cell andadministered to a patient for the treatment or prevention of cancers.Experiments shown in FIG. 12 -FIG. 13 support the validity of thisapproach.

CARs that contain an NME7 fragment in its extracellular domain aretransfected or transduced into an immune system cell, typically a Tcell, and administered to a patient for the treatment or prevention ofcancers. In one aspect, the cancer is a MUC1*-positive cancer. Inanother aspect, the cancer is a metastatic cancer.

Agents that inhibit an enzyme that cleaves NME7 can be used to treat orprevent cancers. Some forms of NME7 are sequestered within the cell andtherefore are not secreted from the cell whereupon they can act asgrowth factors to promote cancers. Full-length NME7 is 42 kDa. However,we found that a ˜33kDaNME7 species that is devoid of the DM10 domain andappears to be essentially identical to the recombinant NME7_(AB) that wegenerated, is secreted from cancer cells and stem cells. This ˜33 kDaNME7 species and another ˜25 kDa NME7 species may be cleavage productsthat would be eliminated by an agent that inhibited cleavage of NME7.

The detection of elevated levels of NME7, or an ˜33 kDa NME7 species,which we call NME7_(AB)-like species, or NME7-X1 in a patient sample isdiagnostic of the presence of cancer or its progression to a moreaggressive or metastatic state. The inventors have discovered that bothearly stage, naïve stem cells and cancer cells, especiallyMUC1*-positive cancer cells, express high levels of a ˜33 kDa NME7 thatis devoid of the DM10 domain and NME7-X1.

NME7-X1 was recently listed in a protein database as being a theoreticalalternative isoform of NME7, however, it had never been detected intissues or cells. We designed primers that differentiate NME7-X1 fromNME7 by PCR. The expression levels of human NME7, NME7a, NME7b andNME7-X1 were measured by PCR in a panel of cells that includedfibroblast cells, human embryonic stem cells, human iPS cells, T47Dhuman breast cancer cells, DU145 human prostate cancer cells, PC3 humanprostate cancer cells, HEK295 human fetal liver cells, and other humanstem cell lines. NME7 is expressed at higher levels in cancer cells thanin stem cells. Particularly, NME7-X1 is expressed 10-fold higher inprostate cancer cells and 3-fold higher in breast cancer cells, than itis in fibroblast cells or stem cells. NME7-X1 is expressed ˜5-foldhigher in HEK293 fetal liver cells than it is in fibroblast cells orstem cells and therefore predicts that NME7-X1 is elevated in livercancers. NME7b is expressed 17-25-times higher in prostate cancer cellsthan in stem cells.

Detection of elevated levels of NME7 species in a patient sample will beindicators that the patient has a cancer or is at risk of developing acancer. Levels of NME7 species levels can be measured or assessed byPCR, hybridization schemes, cycling probe technologies, FISH,immunocytochemistry, IHC, Western blot, immunoprecipitation, sandwichassays, ELISA assays and the like. The patient sample may be a fluidsample, a blood sample, milk, urine, cells, liquid biopsy, biopsy andthe like. In a patient diagnosed with cancer, elevated levels of NME7species are indicators of increased metastatic potential. Elevatedlevels of NME7-X1 are indicators of prostate cancer. Antibodies of theinvention are used to detect and distinguish NME7 species and are usedas a diagnostic tool.

Because adult cells and tissues do not express significant levels ofNME7 or secrete NME7, an effective way to diagnose cancer or to diagnosea more aggressive or metastatic form, or a shift to a more aggressiveform, is to measure levels of NME7 in a sample from a patient, from acollection of cells or tissues or from cultured cells, compared to NME7levels in a healthy sample or compared to levels of NME7 known to existin healthy adult cells or tissues. Increased levels of NME7 indicate thepresence of cancer, the presence of a metastatic cancer or the onset ofmetastasis. Increased levels of NME7 is also indicative of aMUC1*-positive cancer. The sample assayed for the presence of NME7 maybe a collection of cells that may be cultured cell lines or cells from apatient, a bodily fluid, a blood sample, a tissue specimen, or a biopsyspecimen. Therefore, a diagnostic assay that will detect the presence ofcancer or the progression of cancer, comprises the steps of: 1)obtaining a sample from a patient having cancer or at risk of developinga cancer; 2) subjecting that sample to an assay capable of detecting ormeasuring levels of NME7, or levels of nucleic acids encoding NME7; 3)comparing levels of the measured NME7 protein or NME7-encoding nucleicacids in the test sample to levels in control patients or control cells;4) determining that the levels of NME7 or nucleic acids encoding NME7are elevated compared to the controls; and 5) concluding that the donorof the test sample has cancer or has had a progression of cancer if thecontrol to which the test was compared came from a donor previouslydiagnosed with a cancer.

In this assay, the control sample to which the test sample is comparedcan be non-cancerous cells, cultured cells, a sample from a healthydonor, a non-cancerous sample from the donor, or a sample from the donorof the test sample wherein the control sample was taken from the donorat a previous point in time. The source of such samples may be anyspecimen taken from the patient being tested for the presence orprogression of cancer, including bodily fluids, cerebrospinal fluid,bone marrow samples, blood, tissues, cells, biopsy tissues or cells,cultured cells derived from a patient's cells and the like. The sourceof the sample to which the test sample is compared can be bodily fluids,cerebrospinal fluid, bone marrow samples, blood, tissues, cells, biopsytissues or cells, or cultured cells that may be derived from a healthydonor or the test patient wherein the samples were taken at a previouspoint in time. The measured levels to which the test sample is comparedmay be from previously recorded data and compiled into lists forcomparison to test samples.

Theranostics

Patients diagnosed with elevated levels of NME7 protein or nucleic acidsencoding NME7 are then treated with therapeutic agents that suppressexpression of NME7, inhibit cleavage of NME7 or inhibit NME7 binding toits targets, wherein such interaction promotes cancers. An importanttarget of NME7 or a cleavage product of NME7, is MUC1*. NME7 binds toand dimerizes the extracellular domain of MUC1*. Therefore, patientsdiagnosed with elevated levels of NME7 will benefit from treatment withtherapeutic agents that inhibit NME7 and/or therapeutic agents thatinhibit the dimerization of a cleaved form of MUC1, whose extracellulardomain is comprised of some or all of the PSMGFR sequence. Thusassessing suitability of cancer treatments and administration of aneffective amount of a therapeutic for the treatment or prevention ofcancers would consists of the steps of: 1) obtaining a sample from apatient suspected of having a cancer or at risk of developing a canceror at risk of developing a metastatic cancer; 2) measuring an amount ofNME7 or a cleavage product thereof or an NME7 encoding nucleic acidwherein the measured levels are significantly above those measured in acontrol sample; 3) determining that the patient has a cancer or hasdeveloped a more aggressive or a metastatic cancer; 4) administering tothe patient an effective amount of a therapeutic agent that suppressesexpression of NME7, inhibits cleavage of NME7 or inhibits NME7 bindingto its targets and/or administering to the patient an effective amountof a therapeutic agent that suppresses expression of MUC1, inhibitscleavage of MUC1 to MUC1* or inhibits MUC1* binding to its targets. In apreferred embodiment, the therapeutic agent that inhibits NME7 bindingto its targets, inhibits its interaction with MUC1*. In a more preferredembodiments, it inhibits its interaction with the extracellular domainof MUC1* comprised essentially of the PSMGFR sequence. In a preferredembodiment, the therapeutic agent that inhibits MUC1* binding to itstargets, inhibits the interaction between MUC1* and NME7. In a morepreferred embodiment, the therapeutic agent that inhibits theinteraction between MUC1* and NME7 inhibits the binding of MUC1* to theportion of NME7 that is comprised essentially of the sequence of NME7&B.

Chemically Modified Peptides

Polypeptide or antibody therapeutics may suffer from short circulatinghalf-life, and proteolytic degradation and low solubility. To improvethe pharmacokinetics and pharmacodynamics properties of the inventivebiopharmaceuticals, methods such as manipulation of the amino acidsequence may be made to decrease or increase immunogenicity and decreaseproteolytic cleavage; fusion or conjugation of the peptides toimmunoglobulins and serum proteins, such as albumin may be made;incorporation into drug delivery vehicles for the biopharmaceuticalssuch as the inventive peptides and antibodies for protection and slowrelease may also be made; and conjugating to natural or syntheticpolymers are also contemplated. In particular, for synthetic polymerconjugation, pegylation or acylation, such as N-acylation, S-acylationand so forth are also contemplated.

Nucleic Acid Constructs

Also provided is an expression vector comprising a nucleic acid moleculeof the invention as described herein, wherein the nucleic acid moleculeis operatively linked to an expression control sequence. Also providedis a host-vector system for the production of a polypeptide whichcomprises the expression vector of the invention which has beenintroduced into a host cell suitable for expression of the polypeptide.The suitable host cell may be a bacterial cell such as E. coli, a yeastcell, such as Pichia pastoris, an insect cell, such as Spodopterafrugiperda, or a mammalian cell, such as a COS, HEK or CHO cell.

The present invention also provides for methods of producing thepolypeptides of the invention by growing cells of the host-vector systemdescribed herein, under conditions permitting production of thepolypeptide and recovering the polypeptide so produced. The polypeptidesuseful for practicing the present invention may be prepared byexpression in a prokaryotic or eukaryotic expression system.

The recombinant gene may be expressed and the polypeptide purifiedutilizing any number of methods. The gene may be subcloned into abacterial expression vector, such as for example, but not by way oflimitation, pZErO.

The polypeptides may be purified by any technique which allows for thesubsequent formation of a stable, biologically active protein. Forexample, and not by way of limitation, the factors may be recovered fromcells either as soluble proteins or as inclusion bodies, from which theymay be extracted quantitatively by 8M guanidinium hydrochloride anddialysis. In order to further purify the factors, any number ofpurification methods may be used, including but not limited toconventional ion exchange chromatography, affinity chromatography,different sugar chromatography, hydrophobic interaction chromatography,reverse phase chromatography or gel filtration.

When used herein, polypeptide includes functionally equivalent moleculesin which amino acid residues are substituted for residues within thesequence resulting in a silent or conservative change. For example, oneor more amino acid residues within the sequence can be substituted byanother amino acid of a similar polarity, which acts as a functionalequivalent, resulting in a silent or conservative alteration.Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. The potential glycosylation amino acids include serine,threonine, and asparagine. Also included within the scope of theinvention are proteins or fragments or derivatives thereof which exhibitthe same or similar biological activity and derivatives which aredifferentially modified during or after translation, e.g., byglycosylation, proteolytic cleavage, linkage to an antibody molecule orother cellular ligand, etc.

Any of the methods known to one skilled in the art for the insertion ofDNA fragments into a vector may be used to construct expression vectorsencoding the polypeptides of the invention using appropriatetranscriptional/translational control signals and protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinations (genetic recombination).Expression of nucleic acid sequence encoding the polypeptides of theinvention may be regulated by a second nucleic acid sequence so that thepolypeptide is expressed in a host transformed with the recombinant DNAmolecule. For example, expression of the polypeptides described hereinmay be controlled by any promoter/enhancer element known in the art.Promoters which may be used to control expression of the polypeptideinclude, but are not limited to the long terminal repeat as described inSquinto et al., (1991, Cell 65:1-20); the SV40 early promoter region(Bernoist and Chambon, 1981, Nature 290:304-310), the CMV promoter, theM-MuLV 5′ terminal repeat the promoter contained in the 3′long terminalrepeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797),the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl.Acad. Sci. U.S.A. 78:144-1445), the regulatory sequences of themetallothionein gene (Brinster et al., 1982, Nature 296:39-42);prokaryotic expression vectors such as the β-lactamase promoter(Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25), see also “Useful proteins from recombinantbacteria” in Scientific American, 1980, 242:74-94; promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADH (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter, and the following animal transcriptional controlregions, which exhibit tissue specificity and have been utilized intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz etal., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald,1987, Hepatology 7:425-515); insulin gene control region which is activein pancreatic beta cells (Hanahan, 1985, Nature 315:115-122),immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444),mouse mammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495),Sendai virus, lenti virus, albumin gene control region which is activein liver (Pinkert et al., 1987, Genes and Devel. 1:268-276),alpha-fetoprotein gene control region which is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science235:53-58); alpha 1-antitrypsin gene control region which is active inthe liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globingene control region which is active in myeloid cells (Mogram et al.,1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); myelinbasic protein gene control region which is active in oligodendrocytecells in the brain (Readhead et al., 1987, Cell 48:703-712); myosinlight chain-2 gene control region which is active in skeletal muscle(Shani, 1985, Nature 314:283-286), and gonadotropic releasing hormonegene control region which is active in the hypothalamus (Mason et al.,1986, Science 234:1372-1378).

Thus, according to the invention, expression vectors capable of beingreplicated in a bacterial or eukaryotic host comprising nucleic acidsencoding a polypeptide as described herein, are used to transfect thehost and thereby direct expression of such nucleic acid to producepolypeptides which may then be recovered in biologically active form. Asused herein, a biologically active form includes a form capable ofbinding to the relevant receptor and causing a differentiated functionand/or influencing the phenotype of the cell expressing the receptor.

Expression vectors containing the nucleic acid inserts can be identifiedby without limitation, at least three general approaches: (a) DNA-DNAhybridization, (b) presence or absence of “marker” gene functions, and(c) expression of inserted sequences. In the first approach, thepresence of foreign nucleic acids inserted in an expression vector canbe detected by DNA-DNA hybridization using probes comprising sequencesthat are homologous to an inserted nucleic acid sequences. In the secondapproach, the recombinant vector/host system can be identified andselected based upon the presence or absence of certain “marker” genefunctions (e.g., thymidine kinase activity, resistance to antibiotics,transformation phenotype, occlusion body formation in baculovirus, etc.)caused by the insertion of foreign nucleic acid sequences in the vector.For example, if an eft nucleic acid sequence is inserted within themarker gene sequence of the vector, recombinants containing the insertcan be identified by the absence of the marker gene function. In thethird approach, recombinant expression vectors can be identified byassaying the foreign nucleic acid product expressed by the recombinantconstructs. Such assays can be based, for example, on the physical orfunctional properties of the nucleic acid product of interest, forexample, by binding of a ligand to a receptor or portion thereof whichmay be tagged with, for example, a detectable antibody or portionthereof or binding to antibodies produced against the protein ofinterest or a portion thereof.

The polypeptide, in particular modified of the present invention, may beexpressed in the host cells transiently, constitutively or permanently.

Effective doses useful for treating the diseases or disorders indicatedin the present application may be determined using methods known to oneskilled in the art (see, for example, Fingl, et al., The PharmacologicalBasis of Therapeutics, Goodman and Gilman, eds. Macmillan Publishing Co,New York, pp. 1-46 (1975). Pharmaceutical compositions for use accordingto the invention include the polypeptides described above in apharmacologically acceptable liquid, solid or semi-solid carrier, linkedto a carrier or targeting molecule (e.g., antibody, hormone, growthfactor, etc.) and/or incorporated into liposomes, microcapsules, andcontrolled release preparation prior to administration in vivo. Forexample, the pharmaceutical composition may comprise a polypeptide in anaqueous solution, such as sterile water, saline, phosphate buffer ordextrose solution. Alternatively, the active agents may be comprised ina solid (e.g. wax) or semi-solid (e.g. gelatinous) formulation that maybe implanted into a patient in need of such treatment. Theadministration route may be any mode of administration known in the art,including but not limited to intravenously, intrathecally,subcutaneously, intrauterinely, by injection into involved tissue,intraarterially, intranasally, orally, or via an implanted device.

Administration may result in the distribution of the active agent of theinvention throughout the body or in a localized area. For example, insome conditions, which involve distant regions of the nervous system,intravenous or intrathecal administration of agent may be desirable. Insome situations, an implant containing active agent may be placed in ornear the lesioned area. Suitable implants include, but are not limitedto, gelfoam, wax, spray, or microparticle-based implants.

The present invention also provides for pharmaceutical compositionscomprising the polypeptides described herein, in a pharmacologicallyacceptable vehicle. The compositions may be administered systemically orlocally. Any appropriate mode of administration known in the art may beused, including, but not limited to, intravenous, intrathecal,intraarterial, intranasal, oral, subcutaneous, intraperitoneal, or bylocal injection or surgical implant. Sustained release formulations arealso provided for.

Gene Therapy

Gene therapy refers to therapy performed by the administration to asubject of an expressed or expressible nucleic acid. In this embodimentof the invention, the nucleic acids produce their encoded protein thatmediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, N Y (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors, or by direct injection of naked DNA,or coating with lipids or cell-surface receptors or transfecting agents,encapsulation in liposomes, microparticles, or microcapsules, or byadministering them in linkage to a peptide which is known to enter thenucleus, by administering it in linkage to a ligand subject toreceptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem.262:4429-4432 (1987)) (which can be used to target cell typesspecifically expressing the receptors) and so on. In another embodiment,nucleic acid-ligand complexes can be formed in which the ligandcomprises a fusogenic viral peptide to disrupt endosomes, allowing thenucleic acid to avoid lysosomal degradation. In yet another embodiment,the nucleic acid can be targeted in vivo for cell specific uptake andexpression, by targeting a specific receptor. Alternatively, the nucleicacid can be introduced intracellularly and incorporated within host cellDNA for expression, by homologous recombination (Koller and Smithies,Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding the polypeptide are used. The nucleic acid sequencesencoding the polypeptide to be used in gene therapy are cloned into oneor more vectors, which facilitates delivery of the gene into a patient.Lentiviral vectors, such as retroviral vectors, and other vectors suchas adenoviral vectors and adeno-associated viruses are examples of viralvectors that may be used. Retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA.

Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia because they naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. In addition, adeno-associatedvirus (AAV) has also been proposed for use in gene therapy.

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion andso on. Numerous techniques are known in the art for the introduction offoreign genes into cells and may be used in accordance with the presentinvention, provided that the necessary developmental and physiologicalfunctions of the recipient cells are not disrupted. The technique shouldprovide for the stable transfer of the nucleic acid to the cell, so thatthe nucleic acid is expressible by the cell and preferably heritable andexpressible by its cell progeny.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asT-lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, and so on.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding the polypeptide are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention.

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Therapeutic Composition

The formulation of therapeutic compounds is generally known in the artand reference can conveniently be made to Remington's PharmaceuticalSciences, 17th ed., Mack Publishing Co., Easton, Pa., USA. For example,from about 0.05 ng to about 20 mg per kilogram of body weight per daymay be administered. Dosage regime may be adjusted to provide theoptimum therapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. The activecompound may be administered in a convenient manner such as by the oral,intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intra ocular, intradermal or suppository routes or implanting (egusing slow release molecules by the intraperitoneal route or by usingcells e.g. monocytes or dendrite cells sensitized in vitro andadoptively transferred to the recipient). Depending on the route ofadministration, the peptide may be required to be coated in a materialto protect it from the action of enzymes, acids and other naturalconditions which may inactivate said ingredients.

For example, the low lipophilicity of the peptides will allow them to bedestroyed in the gastrointestinal tract by enzymes capable of cleavingpeptide bonds and in the stomach by acid hydrolysis. In order toadminister peptides by other than parenteral administration, they willbe coated by, or administered with, a material to prevent itsinactivation. For example, peptides may be administered in an adjuvant,co-administered with enzyme inhibitors or in liposomes. Adjuvantscontemplated herein include resorcinols, non-ionic surfactants such aspolyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzymeinhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DEP) and trasylol. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes.

The active compounds may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propylene glycoland liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsuperfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, chlorobutanol, phenol, sorbic acid, theomersal and the like. Inmany cases, it will be preferable to include isotonic agents, forexample, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecomposition of agents delaying absorption, for example, aluminiummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterile active ingredient into a sterile vehicle which containsthe basic dispersion medium and the required other ingredients fromthose enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze-drying technique whichyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof

When the peptides are suitably protected as described above, the activecompound may be orally administered, for example, with an inert diluentor with an assimilable edible carrier, or it may be enclosed in hard orsoft shell gelatin capsule, or it may be compressed into tablets, or itmay be incorporated directly with the food of the diet. For oraltherapeutic administration, the active compound may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 1% by weightof active compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 5 toabout 80% of the weight of the unit. The amount of active compound insuch therapeutically useful compositions is such that a suitable dosagewill be obtained. Preferred compositions or preparations according tothe present invention are prepared so that an oral dosage unit formcontains between about 0.1 μg and 2000 mg of active compound.

The tablets, pills, capsules and the like may also contain thefollowing: A binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin may be added or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compound,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound may be incorporated intosustained-release preparations and formulations.

Delivery Systems

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis, construction of a nucleicacid as part of a retroviral or other vector, etc. Methods ofintroduction include but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, intra ocular,epidural, and oral routes. The compounds or compositions may beadministered by any convenient route, for example by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local. In addition, it may bedesirable to introduce the pharmaceutical compounds or compositions ofthe invention into the central nervous system by any suitable route,including intraventricular and intrathecal injection; intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir, such as an Ommaya reservoir. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody or a peptide of the invention, care must be taken to usematerials to which the protein does not absorb. In another embodiment,the compound or composition can be delivered in a vesicle, in particulara liposome. In yet another embodiment, the compound or composition canbe delivered in a controlled release system. In one embodiment, a pumpmay be used. In another embodiment, polymeric materials can be used. Inyet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, thus requiring only a fraction ofthe systemic dose.

Sequence Listing Free Text

As regards the use of nucleotide symbols other than a, g, c, t, theyfollow the convention set forth in WIPO Standard ST.25, Appendix 2,Table 1, wherein k represents t or g; n represents a, c, t or g; mrepresents a or c; r represents a or g; s represents c or g; wrepresents a or t and y represents c or t.

MTPGTQSPFF LLLLLTVLTV VTGSGHASST PGGEKETSAT QRSSVPSSTE KNAVSMTSSVLSSHSPGSGS STTQGQDVTL APATEPASGS AATWGQDVTS VPVTRPALGS TTPPAHDVTSAPDNKPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTSAPDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS APDNRPALGS TAPPVHNVTSASGSASGSAS TLVHNGTSAR ATTTPASKST PFSIPSHHSD TPTTLASHST KTDASSTHHSSVPPLTSSNH STSPQLSTGV SFFFLSFHIS NLQFNSSLED PSTDYYQELQ RDISEMFLQIYKQGGFLGLS NIKFRPGSVV VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVSVSDVPFPFSA QSGAGVPGWG IALLVLVCVL VALAIVYLIA LAVCQCRRKN YGQLDIFPARDTYHPMSEYP TYHTHGRYVP PSSTDRSPYE KVSAGNGGSS LSYTNPAVAA ASANL (SEQ IDNO:1) describes full-length MUC1 Receptor (Mucin 1 precursor, GenbankAccession number: P15941).

(SEQ ID NO: 2) MTPGTQSPFFLLLLLTVLT (SEQ ID NO: 3)MTPGTQSPFFLLLLLTVLT VVTA (SEQ ID NO: 4) MTPGTQSPFFLLLLLTVLT VVTG

SEQ ID NOS:2, 3 and 4 describe N-terminal MUC-1 signaling sequence fordirecting MUC1 receptor and truncated isoforms to cell membrane surface.Up to 3 amino acid residues may be absent at C-terminal end as indicatedby variants in SEQ ID NOS:2, 3 and 4.

GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANL (SEQ ID NO:5) describes atruncated MUC1 receptor isoform having nat-PSMGFR at its N-terminus andincluding the transmembrane and cytoplasmic sequences of a full-lengthMUC1 receptor.

GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:6) describesthe extracellular domain of Native Primary Sequence of the MUC1 GrowthFactor Receptor (nat-PSMGFR—an example of “PSMGFR”):TINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:7) describes theextracellular domain of Native Primary Sequence of the MUC1 GrowthFactor Receptor (nat-PSMGFR—An example of “PSMGFR”), having a singleamino acid deletion at the N-terminus of SEQ ID NO: 6).

GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:8) describesthe extracellular domain of “SPY” functional variant of the nativePrimary Sequence of the MUC1 Growth Factor Receptor having enhancedstability (var-PSMGFR—An example of “PSMGFR”).

TINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:9) describes theextracellular domain of “SPY” functional variant of the native PrimarySequence of the MUC1 Growth Factor Receptor having enhanced stability(var-PSMGFR—An example of “PSMGFR”), having a single amino acid deletionat the C-terminus of SEQ ID NO:8).

(SEQ ID NO: 10)tgtcagtgccgccgaaagaactacgggcagctggacatctttccagcccgggatacctaccatcctatgagcgagtaccccacctaccacacccatgggcgctatgtgccccctagcagtaccgatcgtagcccctatgagaaggtttctgcaggtaacggtggcagcagcctctcttacacaaacccagcagtggcagccgcttctgccaacttgdescribes MUC1 cytoplasmic domain nucleotide sequence. (SEQ ID NO: 11)CQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANLdescribes MUC1 cytoplasmic domain amino acid sequence. (SEQ ID NO: 12)gagatcctgagacaatgaatcatagtgaaagattcgttttcattgcagagtggtatgatccaaatgcttcacttcttcgacgttatgagcttttattttacccaggggatggatctgttgaaatgcatgatgtaaagaatcatcgcacctttttaaagcggaccaaatatgataacctgcacttggaagatttatttataggcaacaaagtgaatgtcttttctcgacaactggtattaattgactatggggatcaatatacagctcgccagctgggcagtaggaaagaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggtatgttgaatacactatattcagtacattttgttaataggagagcaatgtttattttcttgatgtactttatgtatagaaaataadescribes NME7 nucleotide sequence (NME7: GENBANK ACCESSION AB209049).(SEQ ID NO: 13) DPETMNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGENIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGMLNTLYSVHFVNRRAMFIFLMYFMYRKdescribes NME7 amino acid sequence (NME7: GENBANK ACCESSION AB209049).(SEQ ID NO: 14)atggtgctactgtctactttagggatcgtctttcaaggcgaggggcctcctatctcaagctgtgatacaggaaccatggccaactgtgagcgtaccttcattgcgatcaaaccagatggggtccagcggggtcttgtgggagagattatcaagcgttttgagcagaaaggattccgccttgttggtctgaaattcatgcaagcttccgaagatcttctcaaggaacactacgttgacctgaaggaccgtccattctttgccggcctggtgaaatacatgcactcagggccggtagttgccatggtctgggaggggctgaatgtggtgaagacgggccgagtcatgctcggggagaccaaccctgcagactccaagcctgggaccatccgtggagacttctgcatacaagttggcaggaacattatacatggcagtgattctgtggagagtgcagagaaggagatcggcttgtggtttcaccctgaggaactggtagattacacgagctgtgctcagaactggatctatgaatgadescribes NM23-H1 nucleotide sequence (NM23-H1: GENBANK ACCESSION AF487339).(SEQ ID NO: 15) MVLLSTLGIVFQGEGPPISSCDTGTMANCERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVGLKFMQASEDLLKEHYVDLKDRPFFAGLVKYMHSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGSDSVESAEKEIGLWFHPEEL VDYTSCAQNWIYENM23-H1 describes amino acid sequence (NM23-H1: GENBANK ACCESSION AF487339).(SEQ ID NO: 16)atggtgctactgtctactttagggatcgtctttcaaggcgaggggcctcctatctcaagctgtgatacaggaaccatggccaactgtgagcgtaccttcattgcgatcaaaccagatggggtccagcggggtcttgtgggagagattatcaagcgttttgagcagaaaggattccgccttgttggtctgaaattcatgcaagcttccgaagatcttctcaaggaacactacgttgacctgaaggaccgtccattctttgccggcctggtgaaatacatgcactcagggccggtagttgccatggtctgggaggggctgaatgtggtgaagacgggccgagtcatgctcggggagaccaaccctgcagactccaagcctgggaccatccgtggagacttctgcatacaagttggcaggaacattatacatggcggtgattctgtggagagtgcagagaaggagatcggcttgtggtttcaccctgaggaactggtagattacacgagctgtgctcagaactggatctatgaatgadescribes NM23-H1 S120G mutant nucleotide sequence (NM23-H1: GENBANK ACCESSION AF487339).(SEQ ID NO: 17) MVLLSTLGIVFQGEGPPISSCDTGTMANCERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVGLKFMQASEDLLKEHYVDLKDRPFFAGLVKYMHSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGGDSVESAEKEIGLWFHPEEL VDYTSCAQNWIYEdescribes NM23-H1 S120G mutant amino acid sequence (NM23-H1: GENBANK ACCESSION AF487339).(SEQ ID NO: 18)atggccaacctggagcgcaccttcatcgccatcaagccggacggcgtgcagcgcggcctggtgggcgagatcatcaagcgcttcgagcagaagggattccgcctcgtggccatgaagttcctccgggcctctgaagaacacctgaagcagcactacattgacctgaaagaccgaccattcttccctgggctggtgaagtacatgaactcagggccggttgtggccatggtctgggaggggctgaacgtggtgaagacaggccgagtgatgcttggggagaccaatccagcagattcaaagccaggcaccattcgtggggacttctgcattcaggttggcaggaacatcattcatggcagtgattcagtaaaaagtgctgaaaaagaaatcagcctatggtttaagcctgaagaactggttgactacaagtcttgtgctcatgactgggtctatgaataadescribes NM23-H2 nucleotide sequence (NM23-H2: GENBANK ACCESSION AK313448).(SEQ ID NO: 19) MANLERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVAMKFLRASEEHLKQHYIDLKDRPFFPGLVKYMNSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGSDSVKSAEKEISLWFKPEELVDYKSCAHDWVYEdescribes NM23-H2 amino acid sequence (NM23-H2: GENBANK ACCESSION AK313448).Human NM23-H7-2 sequence optimized for E. coli expression: (DNA)(SEQ ID NO: 20)atgcatgacgttaaaaatcaccgtacctttctgaaacgcacgaaatatgataatctgcatctggaagacctgtttattggcaacaaagtcaatgtgttctctcgtcagctggtgctgatcgattatggcgaccagtacaccgcgcgtcaactgggtagtcgcaaagaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattegcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttcaaaattctggataattga (amino acids)(SEQ ID NO: 21) MHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN-Human NME7-A: (DNA) (SEQ ID NO: 22)atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgtttttttga (amino acids)(SEQ ID NO: 23) MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFF- Human NME7-A1: (DNA) (SEQ ID NO: 24)atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttacttga(amino acids) (SEQ ID NO: 25)MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFT- Human NME7-A2: (DNA)(SEQ ID NO: 26)atgaatcatagtgaaagattcgttttcattgcagagtggtatgatccaaatgcttcacttcttcgacgttatgagcttttattttacccaggggatggatctgttgaaatgcatgatgtaaagaatcatcgcacctttttaaagcggaccaaatatgataacctgcacttggaagatttatttataggcaacaaagtgaatgtcttttctcgacaactggtattaattgactatggggatcaatatacagctcgccagctgggcagtaggaaagaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgtttttttga (amino acids) (SEQ ID NO: 27)MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFF- Human NME7-A3:(DNA) (SEQ ID NO: 28)atgaatcatagtgaaagattcgttttcattgcagagtggtatgatccaaatgcttcacttcttcgacgttatgagcttttattttacccaggggatggatctgttgaaatgcatgatgtaaagaatcatcgcacctttttaaagcggaccaaatatgataacctgcacttggaagatttatttataggcaacaaagtgaatgtcttttctcgacaactggtattaattgactatggggatcaatatacagctcgccagctgggcagtaggaaagaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttacttga(amino acids) (SEQ ID NO: 29)MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSS GGCGPANTAKFT-Human NME7-B: (DNA) (SEQ ID NO: 30)atgaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttctga (amino acids)(SEQ ID NO: 31) MNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF- Human NME7-B1: (DNA) (SEQ ID NO: 32)atgaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggataattagtga(amino acids) (SEQ ID NO: 33)MNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN- Human NME7-B2: (DNA)(SEQ ID NO: 34)atgccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttctga (amino acids) (SEQ ID NO: 35)MPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF- Human NME7-B3: (DNA)(SEQ ID NO: 36)atgccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggataattagtga (amino acids) (SEQ ID NO: 37)MPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN-- Human NME7-AB, also known as NME7_(AB): (DNA) (SEQ ID NO: 38)atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggataattagtga (amino acids)(SEQ ID NO: 39) MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFK ILDN--Human NME7-AB1: (DNA) (SEQ ID NO: 40)atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttctga (amino acids) (SEQ ID NO: 41)MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF-Human NME7-A sequence optimized for E. coli expression: (DNA)(SEQ ID NO: 42)atggaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttctga (amino acids)(SEQ ID NO: 43) MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFF-Human NME7-A1 sequence optimized for E. coli expression: (DNA)(SEQ ID NO: 44)atggaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttacctga (amino acids) (SEQ ID NO: 45)MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFT-Human NME7-A2 sequence optimized for E. coli expression: (DNA)(SEQ ID NO: 46)atgaatcactccgaacgctttgtttttatcgccgaatggtatgacccgaatgcttccctgctgcgccgctacgaactgctgttttatccgggcgatggtagcgtggaaatgcatgacgttaaaaatcaccgtacctttctgaaacgcacgaaatatgataatctgcatctggaagacctgtttattggcaacaaagtcaatgtgttctctcgtcagctggtgctgatcgattatggcgaccagtacaccgcgcgtcaactgggtagtcgcaaagaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttctga (amino acids)(SEQ ID NO: 47) MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFF-Human NME7-A3 sequence optimized for E. coli expression: (DNA)(SEQ ID NO: 48)atgaatcactccgaacgctttgtttttatcgccgaatggtatgacccgaatgcttccctgctgcgccgctacgaactgctgttttatccgggcgatggtagcgtggaaatgcatgacgttaaaaatcaccgtacctttctgaaacgcacgaaatatgataatctgcatctggaagacctgtttattggcaacaaagtcaatgtgttctctcgtcagctggtgctgatcgattatggcgaccagtacaccgcgcgtcaactgggtagtcgcaaagaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttacctga (amino acids) (SEQ ID NO: 49)MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSS GGCGPANTAKFT-Human NME7-B sequence optimized for E. coli expression: (DNA)(SEQ ID NO: 50)atgaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttctga(amino acids) (SEQ ID NO: 51)MNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF-Human NME7-B1 sequence optimized for E. coli expression: (DNA)(SEQ ID NO: 52)atgaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttcaaaattctggataattga(amino acids) (SEQ ID NO: 53)MNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN-Human NME7-B2 sequence optimized for E. coli expression: (DNA)(SEQ ID NO: 54)atgccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttctga (amino acids) (SEQ ID NO: 55)MPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF-Human NME7-B3 sequence optimized for E. coli expression: (DNA)(SEQ ID NO: 56)atgccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttcaaaattctggataattga (amino acids) (SEQ ID NO: 57)MPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN-Human NME7-AB, also known as NME7_(AB) sequence optimized for E. coli expression:(DNA) (SEQ ID NO: 58)atggaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttcaaaattctggataattga(amino acids) (SEQ ID NO: 59)MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFK ILDN-Human NME7-AB1, also known as NME7_(AB)1 sequence optimized for E. coli expression:(DNA) (SEQ ID NO: 60)Atggaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttctga (amino acids)(SEQ ID NO: 61) MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFF- Mouse NME6(DNA) (SEQ ID NO: 62)Atgacctccatcttgcgaagtccccaagctcttcagctcacactagccctgatcaagcctgatgcagttgcccacccactgatcctggaggctgttcatcagcagattctgagcaacaagttcctcattgtacgaacgagggaactgcagtggaagctggaggactgccggaggttttaccgagagcatgaagggcgttttttctatcagcggctggtggagttcatgacaagtgggccaatccgagcctatatccttgcccacaaagatgccatccaactttggaggacactgatgggacccaccagagtatttcgagcacgctatatagccccagattcaattcgtggaagtttgggcctcactgacacccgaaatactacccatggctcagactccgtggtttccgccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaggaaccccagctgcggtgtggtcctgtgcactacagtccagaggaaggtatccactgtgcagctgaaacaggaggccacaaacaacctaacaaaacctag (amino acids)(SEQ ID NO: 63) MTSILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRTRELQWKLEDCRRFYREHEGRFFYQRLVEFMTSGPIRAYILAHKDAIQLWRTLMGPTRVFRARYIAPDSIRGSLGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPVHYSPEEGIHCAAETGGHKQPNKT- Human NME6: (DNA) (SEQ ID NO: 64)Atgacccagaatctggggagtgagatggcctcaatcttgcgaagccctcaggctctccagctcactctagccctgatcaagcctgacgcagtcgcccatccactgattctggaggctgttcatcagcagattctaagcaacaagttcctgattgtacgaatgagagaactactgtggagaaaggaagattgccagaggttttaccgagagcatgaagggcgttttttctatcagaggctggtggagttcatggccagcgggccaatccgagcctacatccttgcccacaaggatgccatccagctctggaggacgctcatgggacccaccagagtgttccgagcacgccatgtggccccagattctatccgtgggagtttcggcctcactgacacccgcaacaccacccatggttcggactctgtggtttcagccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaagagccccagttgcgctgtggccctgtgtgctatagcccagagggaggtgtccactatgtagctggaacaggaggcctaggaccagcctga(amino acids) (SEQ ID NO: 65)MTQNLGSEMASILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPVCYSPEGGVHYVAGTGGLGPA- Human NME6 1: (DNA) (SEQ ID NO: 66)Atgacccagaatctggggagtgagatggcctcaatcttgcgaagccctcaggctctccagctcactctagccctgatcaagcctgacgcagtcgcccatccactgattctggaggctgttcatcagcagattctaagcaacaagttcctgattgtacgaatgagagaactactgtggagaaaggaagattgccagaggttttaccgagagcatgaagggcgttttttctatcagaggctggtggagttcatggccagcgggccaatccgagcctacatccttgcccacaaggatgccatccagctctggaggacgctcatgggacccaccagagtgttccgagcacgccatgtggccccagattctatccgtgggagtttcggcctcactgacacccgcaacaccacccatggttcggactctgtggtttcagccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaagagccccagttgcgctgtggccctgtgtga (amino acids) (SEQ ID NO: 67)MTQNLGSEMASILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEE PQLRCGPV-Human NME6 2: (DNA) (SEQ ID NO: 68)Atgctcactctagccctgatcaagcctgacgcagtcgcccatccactgattctggaggctgttcatcagcagattctaagcaacaagttcctgattgtacgaatgagagaactactgtggagaaaggaagattgccagaggttttaccgagagcatgaagggcgttttttctatcagaggctggtggagttcatggccagcgggccaatccgagcctacatccttgcccacaaggatgccatccagctctggaggacgctcatgggacccaccagagtgttccgagcacgccatgtggccccagattctatccgtgggagtttcggcctcactgacacccgcaacaccacccatggttcggactctgtggtttcagccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaagagccccagttgcgctgtggccctgtgtga (amino acids) (SEQ ID NO: 69)MLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPV- Human NME6 3: (DNA)(SEQ ID NO: 70)Atgctcactctagccctgatcaagcctgacgcagtcgcccatccactgattctggaggctgttcatcagcagattctaagcaacaagttcctgattgtacgaatgagagaactactgtggagaaaggaagattgccagaggttttaccgagagcatgaagggcgttttttctatcagaggctggtggagttcatggccagcgggccaatccgagcctacatccttgcccacaaggatgccatccagctctggaggacgctcatgggacccaccagagtgttccgagcacgccatgtggccccagattctatccgtgggagtttcggcctcactgacacccgcaacaccacccatggttcggactctgtggtttcagccagcagagagattgcagccttcttccctgacttcagtgaacagcgctggtatgaggaggaagagccccagttgcgctgtggccctgtgtgctatagcccagagggaggtgtccactatgtagctggaacaggaggcctaggaccagcctga (amino acids) (SEQ ID NO: 71)MLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPVCYSPEGGVHY VAGTGGLGPA-Human NME6 sequence optimized for E. coli expression: (DNA)(SEQ ID NO: 72)Atgacgcaaaatctgggctcggaaatggcaagtatcctgcgctccccgcaagcactgcaactgaccctggctctgatcaaaccggacgctgttgctcatccgctgattctggaagcggtccaccagcaaattctgagcaacaaatttctgatcgtgcgtatgcgcgaactgctgtggcgtaaagaagattgccagcgtttttatcgcgaacatgaaggccgtttcttttatcaacgcctggttgaattcatggcctctggtccgattcgcgcatatatcctggctcacaaagatgcgattcagctgtggcgtaccctgatgggtccgacgcgcgtctttcgtgcacgtcatgtggcaccggactcaatccgtggctcgttcggtctgaccgatacgcgcaataccacgcacggtagcgactctgttgttagtgcgtcccgtgaaatcgcggcctttttcccggacttctccgaacagcgttggtacgaagaagaagaaccgcaactgcgctgtggcccggtctgttattctccggaaggtggtgtccattatgtggcgggcacgggtggtctgggtccggcatga(amino acids) (SEQ ID NO: 73)MTQNLGSEMASILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPVCYSPEGGVHYVAGTGGLGPA-Human NME6 1 sequence optimized for E. coli expression: (DNA)(SEQ ID NO: 74)Atgacgcaaaatctgggctcggaaatggcaagtatcctgcgctccccgcaagcactgcaactgaccctggctctgatcaaaccggacgctgttgctcatccgctgattctggaagcggtccaccagcaaattctgagcaacaaatttctgatcgtgcgtatgcgcgaactgctgtggcgtaaagaagattgccagcgtttttatcgcgaacatgaaggccgtttcttttatcaacgcctggttgaattcatggcctctggtccgattcgcgcatatatcctggctcacaaagatgcgattcagctgtggcgtaccctgatgggtccgacgcgcgtctttcgtgcacgtcatgtggcaccggactcaatccgtggctcgttcggtctgaccgatacgcgcaataccacgcacggtagcgactctgttgttagtgcgtcccgtgaaatcgcggcctttttcccggacttctccgaacagcgttggtacgaagaagaagaaccgcaactgcgctgtggcccggtctga(amino acids) (SEQ ID NO: 75)MTQNLGSEMASILRSPQALQLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEE PQLRCGPV-Human NME6 2 sequence optimized for E. coli expression: (DNA)(SEQ ID NO: 76)Atgctgaccctggctctgatcaaaccggacgctgttgctcatccgctgattctggaagcggtccaccagcaaattctgagcaacaaatttctgatcgtgcgtatgcgcgaactgctgtggcgtaaagaagattgccagcgtttttatcgcgaacatgaaggccgtttcttttatcaacgcctggttgaattcatggcctctggtccgattcgcgcatatatcctggctcacaaagatgcgattcagctgtggcgtaccctgatgggtccgacgcgcgtctttcgtgcacgtcatgtggcaccggactcaatccgtggctcgttcggtctgaccgatacgcgcaataccacgcacggtagcgactctgttgttagtgcgtcccgtgaaatcgcggcctttttcccggacttctccgaacagcgttggtacgaagaagaagaaccgcaactgcgctgtggcccggtctga (amino acids) (SEQ ID NO: 77)MLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPV-Human NME6 3 sequence optimized for E. coli expression: (DNA)(SEQ ID NO: 78)Atgctgaccctggctctgatcaaaccggacgctgttgctcatccgctgattctggaagcggtccaccagcaaattctgagcaacaaatttctgatcgtgcgtatgcgcgaactgctgtggcgtaaagaagattgccagcgtttttatcgcgaacatgaaggccgtttcttttatcaacgcctggttgaattcatggcctctggtccgattcgcgcatatatcctggctcacaaagatgcgattcagctgtggcgtaccctgatgggtccgacgcgcgtctttcgtgcacgtcatgtggcaccggactcaatccgtggctcgttcggtctgaccgatacgcgcaataccacgcacggtagcgactctgttgttagtgcgtcccgtgaaatcgcggcctttttcccggacttctccgaacagcgttggtacgaagaagaagaaccgcaactgcgctgtggcccggtctgttattctccggaaggtggtgtccattatgtggcgggcacgggtggtctgggtccggcatga(amino acids) (SEQ ID NO: 79)MLTLALIKPDAVAHPLILEAVHQQILSNKFLIVRMRELLWRKEDCQRFYREHEGRFFYQRLVEFMASGPIRAYILAHKDAIQLWRTLMGPTRVFRARHVAPDSIRGSFGLTDTRNTTHGSDSVVSASREIAAFFPDFSEQRWYEEEEPQLRCGPVCYSPEGGVHY VAGTGGLGPA-OriGene-NME7-1 full length (DNA) (SEQ ID NO: 80)gacgttgtatacgactcctatagggcggccgggaattcgtcgactggatccggtaccgaggagatctgccgccgcgatcgccatgaatcatagtgaaagattcgttttcattgcagagtggtatgatccaaatgcttcacttcttcgacgttatgagcttttattttacccaggggatggatctgttgaaatgcatgatgtaaagaatcatcgcacctttttaaagcggaccaaatatgataacctgcacttggaagatttatttataggcaacaaagtgaatgtcttctctcgacaactggtattaattgactatggggatcaatatacagctcgccagctgggcagtaggaaagaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggataatacgcgtacgcggccgctcgagcagaaactcatctcagaagaggatctggcagcaaatgatatcctggattacaaggatgacgacgataaggtttaa (amino acids)(SEQ ID NO: 81) MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDNTRTRRLEQKLISEEDLAAN DILDYKDDDDKVAbnova NME7-1 Full length (amino acids) (SEQ ID NO: 82)MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN Abnova Partial NME7-B(amino acids) (SEQ ID NO: 83)DRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKIL Histidine Tag(SEQ ID NO: 84) (ctcgag)caccaccaccaccaccactga Strept II Tag(SEQ ID NO: 85) (accggt)tggagccatcctcagttcgaaaagtaatga N-10 peptide:(SEQ ID NO: 86) QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA C-10 peptide(SEQ ID NO: 87) GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDV (SEQ ID NO: 88)LALIKPDA (SEQ ID NO: 89) MMMLSRKEALDFHVDHQS (SEQ ID NO: 90) ALDFHVDHQS(SEQ ID NO: 91) EILRDDAICEWKRL (SEQ ID NO: 92) FNELIQFITTGP(SEQ ID NO: 93) RDDAICEW (SEQ ID NO: 94) SGVARTDASESIRALFGTDGIRNAA(SEQ ID NO: 95) ELFFPSSGG (SEQ ID NO: 96) KFTNCTCCIVKPHAVSEGLLGKILMA(SEQ ID NO: 97) LMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVT (SEQ ID NO: 98)EFYEVYKGVVTEYHD (SEQ ID NO: 99)EIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNA (SEQ ID NO: 100) YSGPCVAM(SEQ ID NO: 101) FREFCGP (SEQ ID NO: 102) VHCTDLPEDGLLEVQYFFKILDN(SEQ ID NO: 103) IQNAVHCTD (SEQ ID NO: 104) TDLPEDGLLEVQYFFKILDN(SEQ ID NO: 105) PEDGLLEVQYFFK (SEQ ID NO: 106) EIINKAGFTITK(SEQ ID NO: 107) MLSRKEALDFHVDHQS (SEQ ID NO: 108) NELIQFITT(SEQ ID NO: 109) EILRDDAICEWKRL (SEQ ID NO: 110) SGVARTDASESIRALFGTDGI(SEQ ID NO: 111) SGVARTDASES (SEQ ID NO: 112) ALFGTDGI (SEQ ID NO: 113)NCTCCIVKPHAVSE (SEQ ID NO: 114) LGKILMAIRDA (SEQ ID NO: 115)EISAMQMFNMDRVNVE (SEQ ID NO: 116) EVYKGVVT (SEQ ID NO: 117) EYHDMVTE(SEQ ID NO: 118) EFCGPADPEIARHLR (SEQ ID NO: 119) AIFGKTKIQNAV(SEQ ID NO: 120) LPEDGLLEVQYFFKILDN (SEQ ID NO: 121) GPDSFASAAREMELFFPImmunizing peptides derived from human NME7 (SEQ ID NO: 122) ICEWKRL(SEQ ID NO: 123) LGKILMAIRDA (SEQ ID NO: 124) HAVSEGLLGK(SEQ ID NO: 125) VTEMYSGP (SEQ ID NO: 126) NATKTFREF (SEQ ID NO: 127)AIRDAGFEI (SEQ ID NO: 128) AICEWKRLLGPAN (SEQ ID NO: 129) DHQSRPFF(SEQ ID NO: 130) AICEWKRLLGPAN (SEQ ID NO: 131) VDHQSRPF(SEQ ID NO: 132) PDSFAS (SEQ ID NO: 133) KAGEIIEIINKAGFTITKImmunizing peptides derived from human NME1 (SEQ ID NO: 134)MANCERTFIAIKPDGVQRGLVGEIIKRFE (SEQ ID NO: 135) VDLKDRPF (SEQ ID NO: 136)HGSDSVESAEKEIGLWF (SEQ ID NO: 137) ERTFIAIKPDGVQRGLVGEIIKRFE(SEQ ID NO: 138) VDLKDRPFFAGLVKYMHSGPVVAMVWEGLN (SEQ ID NO: 139)NIIHGSDSVESAEKEIGLWFHPEELV (SEQ ID NO: 140) KPDGVQRGLVGEIIImmunizing peptide derived from human NME7, but which does not bind NME1(SEQ ID NO: 141) MLSRKEALDFHVDHQS peptide A1 (SEQ ID NO: 142)SGVARTDASES peptide A2 (SEQ ID NO: 143) DAGFEISAMQMFNMDRVNVE peptide B1(SEQ ID NO: 144) EVYKGVVTEYHDMVTE peptide B2 (SEQ ID NO: 145)AIFGKTKIQNAVHCTDLPEDGLLEVQYFF peptide B3 Human NME7 a (DNA)(SEQ ID NO: 146)atgaatcatagtgaaagattcgttttcattgcagagtggtatgatccaaatgcttcacttcttcgacgttatgagcttttattttacccaggggatggatctgttgaaatgcatgatgtaaagaatcatcgcacctttttaaagcggaccaaatatgataacctgcacttggaagatttatttataggcaacaaagtgaatgtcttttctcgacaactggtattaattgactatggggatcaatatacagctcgccagctgggcagtaggaaagaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggataattag (amino acids)(SEQ ID NO: 147) MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN Human NME7 b (DNA)(SEQ ID NO: 148)atgcatgatgtaaagaatcatcgcacctttttaaagcggaccaaatatgataacctgcacttggaagatttatttataggcaacaaagtgaatgtcttttctcgacaactggtattaattgactatggggatcaatatacagctcgccagctgggcagtaggaaagaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggataattag (amino acids) (SEQ ID NO: 149)MHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDNHuman NME7-AB also known as NME7_(AB) (DNA) (SEQ ID NO: 150)atggaaaaaacgctagccctaattaaaccagatgcaatatcaaaggctggagaaataattgaaataataaacaaagctggatttactataaccaaactcaaaatgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggataattag (amino acids)(SEQ ID NO: 151) MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFK ILDNHuman NME7-X1 (DNA) (SEQ ID NO: 152)atgatgatgctttcaaggaaagaagcattggattttcatgtagatcaccagtcaagaccctttttcaatgagctgatccagtttattacaactggtcctattattgccatggagattttaagagatgatgctatatgtgaatggaaaagactgctgggacctgcaaactctggagtggcacgcacagatgcttctgaaagcattagagccctctttggaacagatggcataagaaatgcagcgcatggccctgattcttttgcttctgcggccagagaaatggagttgttttttccttcaagtggaggttgtgggccggcaaacactgctaaatttactaattgtacctgttgcattgttaaaccccatgctgtcagtgaaggactgttgggaaagatcctgatggctatccgagatgcaggttttgaaatctcagctatgcagatgttcaatatggatcgggttaatgttgaggaattctatgaagtttataaaggagtagtgaccgaatatcatgacatggtgacagaaatgtattctggcccttgtgtagcaatggagattcaacagaataatgctacaaagacatttcgagaattttgtggacctgctgatcctgaaattgcccggcatttacgccctggaactctcagagcaatctttggtaaaactaagatccagaatgctgttcactgtactgatctgccagaggatggcctattagaggttcaatacttcttcaagatcttggataattag (amino acids) (SEQ ID NO: 153)MMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDN*Human NME7 a (optimized for E coli expression) (DNA) (SEQ ID NO: 154)atgaatcactccgaacgctttgtttttatcgccgaatggtatgacccgaatgcttccctgctgcgccgctacgaactgctgttttatccgggcgatggtagcgtggaaatgcatgacgttaaaaatcaccgtacctttctgaaacgcacgaaatatgataatctgcatctggaagacctgtttattggcaacaaagtcaatgtgttctctcgtcagctggtgctgatcgattatggcgaccagtacaccgcgcgtcaactgggtagtcgcaaagaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttcaaaattctggataat(amino acids) (SEQ ID NO: 155)MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDNTGHuman NME7 b (optimized for E coli expression) (DNA) (SEQ ID NO: 156)atgcatgacgttaaaaatcaccgtacctttctgaaacgcacgaaatatgataatctgcatctggaagacctgtttattggcaacaaagtcaatgtgttctctcgtcagctggtgctgatcgattatggcgaccagtacaccgcgcgtcaactgggtagtcgcaaagaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttcaaaattctggataat (amino acids)(SEQ ID NO: 157) MHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILD NTGHuman NME7-AB also known as NME7_(AB) (optimized for E coli expression)(DNA) (SEQ ID NO: 158)atggaaaaaacgctggccctgattaaaccggatgcaatctccaaagctggcgaaattatcgaaattatcaacaaagcgggtttcaccatcacgaaactgaaaatgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttcaaaattctggataat(amino acids) (SEQ ID NO: 159)MEKTLALIKPDAISKAGEIIEIINKAGFTITKLKMMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFK ILDNTGHuman NME7-X1 (optimized for E coli expression) (DNA) (SEQ ID NO: 160)atgatgatgctgagccgtaaagaagccctggattttcatgtcgaccaccagtctcgcccgtttttcaatgaactgattcaattcatcaccacgggtccgattatcgcaatggaaattctgcgtgatgacgctatctgcgaatggaaacgcctgctgggcccggcaaactcaggtgttgcgcgtaccgatgccagtgaatccattcgcgctctgtttggcaccgatggtatccgtaatgcagcacatggtccggactcattcgcatcggcagctcgtgaaatggaactgtttttcccgagctctggcggttgcggtccggcaaacaccgccaaatttaccaattgtacgtgctgtattgtcaaaccgcacgcagtgtcagaaggcctgctgggtaaaattctgatggcaatccgtgatgctggctttgaaatctcggccatgcagatgttcaacatggaccgcgttaacgtcgaagaattctacgaagtttacaaaggcgtggttaccgaatatcacgatatggttacggaaatgtactccggtccgtgcgtcgcgatggaaattcagcaaaacaatgccaccaaaacgtttcgtgaattctgtggtccggcagatccggaaatcgcacgtcatctgcgtccgggtaccctgcgcgcaatttttggtaaaacgaaaatccagaacgctgtgcactgtaccgatctgccggaagacggtctgctggaagttcaatactttttcaaaattctggataat (amino acids)(SEQ ID NO: 161) MMMLSRKEALDFHVDHQSRPFFNELIQFITTGPIIAMEILRDDAICEWKRLLGPANSGVARTDASESIRALFGTDGIRNAAHGPDSFASAAREMELFFPSSGGCGPANTAKFTNCTCCIVKPHAVSEGLLGKILMAIRDAGFEISAMQMFNMDRVNVEEFYEVYKGVVTEYHDMVTEMYSGPCVAMEIQQNNATKTFREFCGPADPEIARHLRPGTLRAIFGKTKIQNAVHCTDLPEDGLLEVQYFFKILDNTG DM10 domain of NME7 (amino acids)(SEQ ID NO: 162) MNHSERFVFIAEWYDPNASLLRRYELLFYPGDGSVEMHDVKNHRTFLKRTKYDNLHLEDLFIGNKVNVFSRQLVLIDYGDQYTARQLGSRKa fragment or variation of PSMGFR peptide (SEQ ID NO: 163)SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY;a fragment or variation of PSMGFR peptide (SEQ ID NO: 164)SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY;a fragment or variation of PSMGFR peptide (SEQ ID NO: 165)VQLTLAFREGTINVHDVETQFNQY; a fragment or variation of PSMGFR peptide(SEQ ID NO: 166) SNIKFRPGSVVVQLTLAFREGTIN;a fragment or variation of PSMGFR peptide (SEQ ID NO: 167)SNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTE;a fragment or variation of PSMGFR peptide (SEQ ID NO: 168)VQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVP.Cys at residue 14 is mutated to Ser of NME7B peptide 3 (B domain):(SEQ ID NO: 169) AIFGKTKIQNAVHSTDLPEDGLLEVQYFF N-10 peptide(SEQ ID NO: 170) QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA C-10 peptide(SEQ ID NO: 171) GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDV

EXAMPLES Example 1—Components of Minimal Serum-Free Base (“MM”) (500mls)

400 ml DME/F12/GlutaMAX I (Invitrogen #10565-018)

100 ml Knockout Serum Replacement (KO-SR, Invitrogen #10828-028)

5 ml 100×MEM Non-essential Amino Acid Solution (Invitrogen #11140-050)

0.9 ml (0.1 mM) β-mercaptoethanol (55 mM stock, Invitrogen #21985-023.

Example 2—Generation of Protein Constructs

For generating recombinant NME7, first, constructs were made to make arecombinant NME7 that could be expressed efficiently and in solubleform. The first approach was to make a construct that would encode thenative NME7 (a) or an alternative splice variant NME7 (b), which has anN-terminal deletion. In some cases, the constructs carried a histidinetag or a strep tag to aid in purification. NME7-a, full-length NME7expressed poorly in E. coli and NME7-b did not express at all in E.coli. However, a novel construct was made in which the DM10 sequence wasdeleted and the NME7 comprised essentially the NDPK A and B domainshaving a calculated molecular weight of 33 kDa.

This novel NME7_(AB) expressed very well in E. coli and existed as thesoluble protein. NME7_(AB) was first purified over an NTA-Ni column andthen further purified by size exclusion chromatography (FPLC) over aSephadex 200 column. Fractions were collected and tested by SDS-PAGE toidentify fractions with the highest and purest expression of NME7_(AB).The FPLC trace for the combined fractions that were the most pure werecombined. The purified NME7_(AB) protein was then tested and shown tofully support the growth of human stem cells and further reverts them tothe most naïve, pre-X-inactivation state. The purified NME7_(AB) wasalso shown to accelerate the growth of cancer cells.

Example 3—ELISA Assay Showing NME7_(AB) Simultaneously Binds to TwoMUC1* Extra Cellular Domain Peptides

Results are shown in FIG. 1 . The PSMGFR peptide bearing a C-terminalCysteine (PSMGFR-Cys) was covalently coupled to BSA using ImjectMaleimide activated BSA kit (Thermo Fisher). PSMGFR-Cys coupled BSA wasdiluted to 10 ug/mL in 0.1M carbonate/bicarbonate buffer pH 9.6 and 50uL was added to each well of a 96 well plate. After overnight incubationat 4° C., the plate was washed twice with PBS-T and a 3% BSA solutionwas added to block remaining binding site on the well. After 1 h at RTthe plate was washed twice with PBS-T and NME7, diluted in PBS-T+1% BSA,was added at different concentrations. After 1 h at RT the plate waswashed 3× with PBS-T and anti-NM23-H7 (B-9, Santa Cruz Biotechnology),diluted in PBS-T+1% BSA, was added at 1/500 dilution. After 1 h at RTthe plate was washed 3× with PBS-T and goat anti mouse-HRP, diluted inPBS-T+1% BSA, was added at 1/3333 dilution. After 1 h at RT the platewas washed 3× with PBS-T and binding of NME7 was measured at 415 nmusing ABTS solution (Pierce).

ELISA MUC1* dimerization: The protocol for NME7 binding was used, andNME7 was used at 11.6 ug/mL.

After 1 h at RT the plate was washed 3× with PBS-T and His-Tagged PSMGFRpeptide (PSMGFR-His) or biotinylated PSMGFR peptide (PSMGFR-biotin),diluted in PBS-T+1% BSA, was added at different concentration. After 1 hat RT the plate was washed 3× with PBS-T and anti-Histag-HRP (Abcam) orstreptavidin-HRP (Pierce), diluted in PBS-T+1% BSA, was added at aconcentration of 1/5000. After 1 h at RT the plate was washed 3× withPBS-T and binding of PSMGFR peptide to NME7 already bound to anotherPSMGFR peptide (which could not signal by anti-His antibody or bystreptavidin) coupled BSA was measured at 415 nm using a ABTS solution(Pierce).

Example 4—Functional Testing of Human Recombinant NME7_(AB)

For testing recombinant NME7_(AB) for ability to maintain pluripotencyand inhibit differentiation, a soluble variant of NME7, NME7_(AB), wasgenerated and purified. Human stem cells (iPS cat #SC101a-1, SystemBiosciences) were grown per the manufacturer's directions in 4 ng/mlbFGF over a layer of mouse fibroblast feeder cells for four passages.These source stem cells were then plated into 6-well cell culture plates(Vita™, Thermo Fisher) that had been coated with 12.5 ug/well of amonoclonal anti-MUC1* antibody, MN-C3. Cells were plated at a density of300,000 cells per well. The base media was Minimal Stem Cell Mediaconsisting of: 400 ml DME/F12/GlutaMAX I (Invitrogen #10565-018), 100 mlKnockout Serum Replacement (KO-SR, Invitrogen #10828-028), 5 ml 100×MEMNon-essential Amino Acid Solution (Invitrogen #11140-050) and 0.9 ml(0.1 mM) β-mercaptoethanol (55 mM stock, Invitrogen #21985-023). Thebase media can be any media. In a preferred embodiment, the base mediais free of other growth factors and cytokines. To the base media wasadded either 8 nM of NME7_(AB) or 8 nM NM23-H1 refolded and purified asstable dimers. Media was changed every 48 hours and due to acceleratedgrowth, had to be harvested and passaged at Day 3 post-plating.Comparable pluripotent stem cell growth was achieved when stem cellswere grown in NM23-H1 dimers or in NME7 monomers.

NME7 and NM23-H1 (NME1) dimers both grew pluripotently and had nodifferentiation even when 100% confluent. As can be seen in the photos,NME7 cells grew faster than the cells grown in NM23-H1 dimers. Cellcounts at the first harvest verified that culture in NME7 produced1.4-times more cells than culture in NM23-H1 dimers. ICC staining forthe typical pluripotent markers confirmed that NME7_(AB) fully supportedhuman stem cell growth, pluripotency, and resisted differentiation.

The NME7 species of ˜30-33 kDa may be an alternative splice isoform or apost translational modification such as cleavage, which may enablesecretion from the cell.

Example 5—Inducing Transition of Cancer Cells to Metastatic Cancer Cellsby Culturing Cells Under Conditions that Revert Stem Cells to a MoreNaïve State

Cancer cells are normally cultured in a serum-containing media such asRPMI. We discovered that culturing cancer cells in the presence ofreagents that make stem cells revert to a more naïve state, makes thecancer cells transform to a more metastatic state.

We demonstrated that NME7_(AB), human NME1 dimers, bacterial NME1dimers, NME7-X1 and “2i” inhibitors were each able to transform regularcancer cells into metastatic cancer cells, which are also called cancerstem cells “CSCs” or tumor initiating cells “TICs”. 2i is the name givento two biochemical inhibitors that researchers found made human stemcells revert to a more naïve state. 2i are MEK and GSK3-beta inhibitorsPD0325901 and CHIR99021, which are added to culture medium to finalconcentrations of about 1 mM and 3 mM, respectively.

NME7_(AB) and NME7-X1 are at a final concentration of about 4 nM whenadded to separate batches of minimal medium to make cancer cellstransform to metastatic cells, although lower and higher concentrationsalso work well in the range of about 1 nM to 16 nM. Human or bacterialNME1 dimers are used at a final concentration of 4 nM to 32 nM, with 16nM typically used in these experiments, wherein the human NME bears theS120G mutation. Lower concentrations may be required if using wild type.It is not intended that these exact concentrations are important. It isimportant that the NME1 proteins are dimers and the range ofconcentrations over which this happens is in the low nanomolar rangealthough certain mutations allow higher concentrations to remain asdimers.

Similarly, the concentrations of NME7 proteins can vary. NME7_(AB) andNME7-X1 are monomers and concentrations used to transform cancer cellsto metastatic cells should allow the proteins to remain as monomers.Various molecular markers have been proposed as being indicators ofmetastatic cancer cells. Different cancer types may have differentmolecules that are up-regulated. For example, the receptor CXCR4 isup-regulated in metastatic breast cancers while E-cadherin, also knownas CHD1, is up-regulated more in metastatic prostate cancers.

In addition to these specific metastasis markers, typical markers ofpluripotency such as OCT4, SOX2, NANOG, and KLF4 are up-regulated ascancers become metastatic. The starting cancer cells and the latermetastatic cancer cells can be assayed by PCR to measure expressionlevels of these genes.

FIG. 2 shows a graph of RT-PCR measurements of T47D breast cancer cellsthat were cultured in a media that contained NME7_(AB). A rho I kinaseinhibitor, ROCi, ROCKi or Ri, was added to prevent the transformed cellsfrom floating off the plate. Expression levels of various metastaticmarkers as well as pluripotent stem cell markers were measured for theparent cells and for the NME7_(AB) cultured cells. The results show thatthe floater cells express higher amounts of metastatic and pluripotencymarkers compared to the cells that received ROCi. We reasoned it wasbecause those measurements were the average of cells that did nottransform and those that did but the ROCi made them remain adherent.This can clearly be seen in figures wherein “—Ri” means adherent cellsthat did not receive ROCi and so were not mixed with the highlymetastatic cells that float.

Prostate cancer cells also transitioned to a more metastatic state whencultured in media containing NM23, aka NME1, or NME7_(AB). Here we showthat for every cell line tested so far, culture in NME7_(AB), human NME1dimers, or bacterial NMEs that have high sequence homology to human,induces transition to a more metastatic state.

FIG. 4 shows a graph of RT-PCR measurements of expression levels ofmetastatic and pluripotency markers for breast cancer cells that arecultured in media containing either 2i inhibitors, NME7_(AB) or both. Ascan be seen, 2i inhibitors are also able to induce the transition ofcancer cells to a more metastatic state. Ovarian cancer cell linesSK-OV3, OV-90, pancreatic cancer cell lines CAPAN-2 and PANC-1, breastcancer cell line MDA-MB all displayed the morphological transition ofgoing from adherent to non-adherent when cultured in NME7_(AB) and or 2iinhibitors.

FIG. 20 shows graphs of RT-PCR measurement of metastatic or pluripotencymarkers for various cancer cell lines cultured for 72 or 144 hours inNME7_(AB). FIG. 20A shows that SK-OV3 cells increase expression ofmetastatic markers CHD1, SOX2 and NME7-X1 when cultured in NME7_(AB).FIG. 20B shows that OV-90 cells increase expression of metastaticmarkers CXCR4 and NME7-X1 after culture in NME7_(AB).

Example 6—Demonstration that Cancer Cells Cultured in NME7 BecomeMetastatic

A functional test of whether or not a population of cancer cells ismetastatic is to implant very low numbers, e.g. 200, of the cells inimmuno-compromised mice and see if they develop into a tumor. Typically5-6 million cancer cells are required to form a tumor in animmuno-compromised mouse. We showed that as few as 50 of the NME-inducedmetastatic cancer cells formed tumors in mice. In addition, mice thatwere injected throughout the test period with human NME7_(AB), NME1, orNME7-X1 developed remote metastases.

T47D human breast cancer cells were cultured in standard RPMI media for14 days with media changes every 48 hours and passed by trypsinizationwhen approximately 75% confluent. The cells were then plated into 6-wellplates and cultured in minimal stem cell media (see Example 1) that wassupplemented with 4 nM NME7_(AB). Media was changed every 48 hours. Byabout Day 4, some cells become detached from the surface and float.Media is carefully changed so as to retain the “floaters” as these arethe cells that have the highest metastatic potential as evidenced byRT-PCR measurement of metastatic markers. On Day 7 or 8, the floatersare harvested and counted. Samples are retained for RT-PCR measurement.The key marker measured is CXCR4 which is up-regulated by 40-200 timesafter being briefly cultured in NME7_(AB).

The freshly harvested floater metastatic cells are xenografted into theflank of female nu/nu athymic mice that have been implanted with 90-dayslow release estrogen pellets. Floater cells were xenografted as 10,000,1,000, 100 or 50 cells each. Half of the mice in each group of 6 werealso injected daily with 32 nM NME7_(AB) near the original implantationsite. The parent T47D cells that were cultured in RPMI media withoutNME7_(AB) were also implanted into mice as 6 million, 10,000 or 100 ascontrols. Mice implanted with the NME7-induced floater cells developedtumors even when as few as 50 cells were implanted. Mice that wereimplanted with the floater cells and that received daily injections ofNME7_(AB) also developed remote tumors or remote metastases in variousorgans. 11 out of the 12 mice, or 92%, that were injected with humanNME7_(AB) after implantation of the NME7_(AB) cultured cancer cells,developed tumors at the injection site. Only 7 out of the 12 mice, or58%, that were not injected with human NME7_(AB) after implantationdeveloped tumors. 9 out of the 11 mice, or 82%, that got tumors and wereinjected with human NME7_(AB) developed multiple tumors remote from theinjection site. None of the mice that were not injected with NME7_(AB)developed multiple, visible tumors.

After sacrifice, RT-PCR and Western blots showed that the remote bumpson the mice injected with NME7_(AB) were indeed human breast tumors.Similar analysis of their organs showed that in addition to remotebumps, mice had randomly metastasized to the liver and lung with humanbreast cancer characteristic of the human breast cancer cells that wereimplanted. As expected, only the mice implanted with 6 million cellsgrew tumors.

Several experiments like the one described above were performed withessentially the same results. In each experiment, there were either 24or 52 mice, including all proper controls.

Example 7—Peptides Selected Because their Sequence is Unique to NME7,A1, A2, B1, B2 and B3, Inhibit the Binding of NME7 Species to MUC1*Extracellular Domain Peptide

NME7 peptides were selected as immunizing agents for antibodyproduction. NME7 peptides A1, A2, B1, B2 and B3 (FIG. 9 ) were chosenusing a process of sequence alignment among human NME1, human NME7 andseveral bacterial NMEs that were homologous to human NME1 or human NME7.Five regions that had high sequence homology among all were identified.However, to prevent selecting peptides that would give rise toantibodies that would inhibit human NME1 as well as human NME7, we choseNME7 sequences that were adjacent to the homologous regions whereinthose peptides had sequences that were different from human NME1. We didELISA assays to see if the peptides on their own could bind to asynthetic MUC1* peptide on the surface and inhibit the binding of humanNME7 or human NME1 to the immobilized peptide (FIG. 11 ). FIG. 11 showsthat the peptides inhibited the binding of NME7 and NME1 to theimmobilized PSMGFR peptide. Recall that each of the NME7 A domain and Bdomain can bind to a PSMGFR peptide. Therefore complete inhibition ofNME7_(AB) binding to a PSMGFR peptide cannot be accomplished with asingle antibody or peptide that is derived from just one domain. Thisshowed that those regions from which the peptides were derived were theregions that interacted with MUC1* and would give rise to antibodiesthat would bind to those regions of NME7 and inhibit its binding toMUC1* receptor.

In another experiment, the free peptides A1, A2, B1, B2 and B3 wereadded to cancer cells in culture that were undergoing transition to amore metastatic state by culturing in either NME7_(AB) or 2i. FIG. 14shows a table of scientist observations when cancer cells are grown ineither NME7_(AB) or 2i inhibitors, and shows that the free peptidesinhibited the morphological change from adherent cells to floaters,which for breast cancer cells is directly correlated to increasedexpression of metastatic markers, especially CXCR4. RT-PCR measurementsconfirm that the NME7_(AB) peptides inhibited the increase in expressionof metastasis marker CXCR4.

FIG. 15 shows a graph of RT-PCR measurements of CXCR4 expression in T47Dbreast cancer cells that were grown in either NME7_(AB) or 2iinhibitors, each of which transform cancer cells to a more metastaticstate, and the inhibitory effect of NME7-derived peptides, A1, A2, B1,B2 and B3, on the metastatic transformation. FIG. 32 shows a table ofrecorded RNA levels in samples that were used for RT-PCR measurement ofCXCR4 in FIG. 15 as well as the threshold cycle number for CXCR4expression as well as for the control housekeeping gene.

Example 8—Anti-NME7 Antibodies Specifically Bind to Human NME7 but notto Human NME1

A standard ELISA assay was performed to determine whether or not theNME7 antibodies we generated by immunization with NME7_(AB) peptides A1,A2, B1, B2, and B3 would bind specifically to NME7_(AB), but not tohuman NME1 as it has healthy functions and it may be detrimental to ahuman to block it with an antibody. The ELISAs of FIG. 24-25 show thatall of the NME7 antibodies that were generated from peptides A1, A2, B1,B2, and B3 bind to human NME7_(AB) (FIG. 24 ) but not to human NME1(FIG. 25 ). The peptides used to generate these antibodies are common toboth NME7_(AB) and NME7-X1. This assays show that the antibodiesgenerated from peptides A1, A2, B1, B2, and B3 specifically bind toNME7_(AB) and by extension will bind to NME7-X1.

NME7A peptide 1 (A domain): (SEQ ID NO: 141) MLSRKEALDFHVDHQSNME7A peptide 2 (A domain): (SEQ ID NO: 142) SGVARTDASESNME7B peptide 1 (B domain): (SEQ ID NO: 143) DAGFEISAMQMFNMDRVNVENME7B peptide 2 (B domain): (SEQ ID NO: 144) EVYKGVVTEYHDMVTENME7B peptide 3 (B domain): (SEQ ID NO: 145)AIFGKTKIQNAVHCTDLPEDGLLEVQYFF

Example 9—Anti-NME7 Specific Antibodies and the Peptides that Generatedthem Inhibit Cancer Cell Growth

Rabbits were immunized with NME7 peptides A1, A2, B1, B2, and B3 andantibodies were generated, collected and purified over a column to whichthe immunizing peptide had been conjugated. T47D breast cancer cellswere plated and cultured according to ATCC protocols in RPMI mediasupplemented with serum. Antibodies generated from immunization withpeptides A1, A2, B1, B2, and B3 were added at the concentrationsindicated in FIG. 12 . Immunizing peptides A1, A2, B1, B2, and B3, andthe PSMGFR extracellular domain peptide of MUC1*, “FLR” here, were alsoadded separately to growing T47D breast cancer cells. Taxol and the E6anti-MUC1* Fab were added as controls. The graph of FIG. 12 shows thatthe antibodies generated, as well as the free peptides, potentlyinhibited the growth of the cancer cells. Note the comparison toinhibition using Taxol, which is a chemotherapy agent that kills healthyand cancer cells alike. Also, for comparison, a polyclonal antibodygenerated using a large stretch of NME7 from amino acid 100 to 376 isshown. Although this antibody is a potent inhibitor of cancer growth itcould have non-specific effects since it can bind to NME1 as well as toNME7.

In a similar experiment, combinations of the antibodies generated fromimmunization with peptides A1, A2, B1, B2, and B3 as well as thepeptides themselves were added to growing cancer cells at theconcentrations indicated. The graphs of cell growth shown in FIG. 13show that the combinations of antibodies and peptides potently inhibitedthe growth of cancer cells. In these two experiments, the cells wereMUC1* positive breast cancer cells.

Example 10—Anti-NME7 Antibodies Inhibit the Transition of Cancer Cellsto Metastatic Cancer Cells

Cancer cells transform to a more metastatic state when cultured in thepresence of agents that revert stem cells to a more naïve state. We havedemonstrated that culturing cancer cells in NME7_(AB), human NME1dimers, bacterial NME1 dimers or MEK and GSK3-beta inhibitors, called“2i”, causes the cells to become more metastatic. As the cellstransition to a more metastatic state, they become non-adherent andfloat off of the culture plate. These floating cells, “floaters” werecollected separately from those that were adherent and were shown to: a)express much higher levels of metastatic genes; and b) when xenograftedinto mice, the floater cells were able to generate tumors when implantedat very low numbers. RT-PCR measurement of specific metastatic markerssuch as CXCR4 in breast cancers, CHD1 in prostate cancer, and otherpluripotent stem cell markers such as OCT4, SOX2, NANOG, KLF4, c-Myc andothers were dramatically over-expressed in cancer cells that werecultured in NME7_(AB) and most over-expressed in the cells that becamenon-adherent, called “floaters” here and in figures.

Here we show that the NME7-specific antibodies, generated byimmunization with NME7-derived peptides A1, A2, B1, B2 and B3, as wellas the peptides themselves, inhibit the transition from cancer cell tometastatic cancer cells. In the first of these experiments, theantibodies generated by immunization with A1, A2, B1, B2 and B3 weretested for their ability to inhibit the metastatic transition induced byculture of T47D breast cancer cells in NME7_(AB) or in 2i inhibitors.The most striking observation was that the antibodies and the peptidesdramatically reduced the number of floater cells, which was the firstindication that the antibodies and peptides had inhibited thetransformation to metastatic cancer cells. In particular, cells to whichthe antibody generated from immunization with the B3 peptide barelygenerated any floater cells.

FIG. 14 shows the recorded observations of the percentage of floatercells visible for each antibody relative to the control wells that didnot receive any antibody treatment. mRNA was extracted from both thefloater cells and the adherent cells. RT-PCR was used to measureexpression levels of metastatic markers, including CXCR4. Treatment withthe anti-NME7 antibodies greatly reduced the amount of metastaticmarkers, such as CXCR4, indicating the antibodies inhibited thetransition to metastatic cancer. (See FIG. 15 ). Notably, the antibodygenerated by immunization with peptide B3, aka antibody #61, essentiallycompletely inhibited the transition to a more metastatic state. FIG. 15Bshows that breast cancer cells that were treated with the NME7_(AB)peptides, A1, A2, B1, B2 and B3, alone were able to potently inhibit thetransition to a more metastatic state induced by culturing the cells ina media containing the 2i inhibitors. Peptide B3 was especiallyeffective as was antibody #61 that it generated. FIG. 15C shows the samegraph but with the Y-axis expanded to show the peptide inhibition ofmetastatic markers. The amount of mRNA, which indicates cell viabilityand growth, was measured. Cells that were treated with antibody had muchless mRNA, indicating that in addition to inhibiting the transition to amore metastatic state, the anti-NME7_(AB) antibodies inhibited thegrowth of the cancer cells. FIG. 16 shows a table of the amounts of RNArecovered for the inhibition experiment shown in FIG. 15A.

Example 11—Anti-NME7 Antibodies Generated with NME7-Derived Peptides A1,A2, B1, B2 and B3 Identify Novel NME7 Species not Detectable Using anyCommercially Available Antibodies

As is known to those skilled in the art, some antibodies recognize alinear portion of the target protein and can be used in Western blotassays while other antibodies recognize a non-linear conformationalmotif and can be used in pull-down or immunoprecipitation assays.Previous to this application, cleaved NME7 or isoform NME7-X1 was notknown to exist. Using antibodies that were commercially available at thetime of filing shows that existing antibodies could not specificallydetect these important NME7 species. B9 (Santa Cruz Biotechnology) is amonoclonal antibody raised against NME7 amino acids 100-376. FIG.19D-19F shows that it only detects full-length 42 kDa NME7. Anothercommercially available antibody, H278, is a rabbit polyclonal raisedagainst NME7 amino acids 100-376, which includes amino acid sequencesthat are not unique to NME7. FIG. 19D-19F shows that this antibody alsostains NME1, which is 17 kDa as well as full-length NME7 and other bandsthat do not appear to be specific to NME7_(AB).

NME7 antibodies generated by immunization with NME7_(AB) peptides A1,A2, B1, B2 or B3 identify new NME7 species including the full-length 42kDa protein, a ˜33kDaNME7 species that may be a cleavage product oralternative isoform, a ˜30 kDa NME7 species that may be a cleavageproduct or alternative isoform, wherein the ˜30 kDa species appears tobe NME7-X1. FIG. 19A-C shows that antibodies generated by peptides A1,B1 and B3 identify the secreted forms of NME7, NME7_(AB) and NME7-X1 ina wide range of cancer cell lines, including T47D breast cancer cells,PC3 and DU145 prostate cancer cells, HEK293 fetal liver cells, andleukemia cells IM-9, K562, and MV411.

Example 12—Generation of Anti-NME7 Antibodies

A synthetic peptide having the sequence of the B3 region of NME7,AIFGKTKIQNAVHCTDLPEDGLLEVQYFFC (SEQ ID NO: 1142), was used to immunizerabbits. Antibodies that resulted from immunization with NME7 peptide B3inhibited the growth of MUC1* positive cancer cells and also inhibitedthe formation of cancer stem cells, which are characterized byupregulation of metastatic markers, ability to grow anchorageindependently, and are able to form tumors in animals from as few as 200cells, whereas regular cancer cells typically require implantation ofabout 4 million cells for tumor engraftment.

In some cases, the NME7 B3 peptide was made with a C14A or C14Vmutation. This sequence more reproducibly generated anti-NME7antibodies.

Monoclonal antibodies were generated in mice according to standardmethods by immunizing with NME7 B3, B3 with C14A mutation, or B3 withC14V mutation. The antibodies listed were selected because of theirability to bind to NME7, NME7-X1, NME7_(AB), but importantly did notbind to NME1, which is thought to be required for some normal cellularfunctions. These antibodies also bind to the NME7 derived peptides B3,B3 with C14A mutation, and B3 with C14V mutation.

Experiments showed that these anti-NME7 antibodies inhibited the bindingof NME7 to the MUC1* extra cellular domain, but did not block thebinding of NME1 to the MUC1* extra cellular domain peptide. Further, theantibodies inhibited the formation of cancer stem cells.

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Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention specifically described herein. Suchequivalents are intended to be encompassed in the scope of the claims.

1.-49. (canceled)
 50. A monoclonal antibody comprising an amino acidsequence in the heavy chain variable region comprising the following: inthe CDR1 region NTFTEYTMH (SEQ ID NO: 388); in the CDR2 regionGFNPNNGVTNYNQKFKG (SEQ ID NO: 389); and in the CDR3 region RYYHSLYVFYFDY(SEQ ID NO: 390); and an amino acid sequence in the light chain variableregion comprising the following: in the CDR1 region SASQGISNYLN (SEQ IDNO:393); in the CDR2 region YTSSLHS (SEQ ID NO: 394); and in the CDR3region QQYSKLPYT (SEQ ID NO: 395).
 51. The monoclonal antibody of claim50, comprising (a) an amino acid sequence in the heavy chain variableregion comprising a sequence of SEQ ID NO: 1139; and (b) an amino acidsequence in the light chain variable region comprising a sequence of anyone of SEQ ID NO:
 1109. 52. The monoclonal antibody of claim 50,comprising (a) an amino acid sequence in the heavy chain variable regioncomprising a sequence of any one of SEQ ID NOs: 1102, 1106, or 1133; and(b) an amino acid sequence in the light chain variable region comprisinga sequence of any one of SEQ ID NOs: 1104, 1108, or
 1135. 53. Themonoclonal antibody of claim 50, which is monovalent.
 54. The monoclonalantibody of claim 50, which is an Fab or a single chain variablefragment antibody (scFv).
 55. The monoclonal antibody of claim 50, whichis bispecific.
 56. A BiTE comprising the antibody of claim
 50. 57. Themonoclonal antibody of claim 50, which is humanized or an engineeredantibody mimic.
 58. An isolated nucleic acid encoding the monoclonalantibody according to claim
 50. 59. A method of preventing or treating aMUC1*-dependent cancer comprising administering to a pharmaceuticalcomposition comprising the antibody of claim
 50. 60. The method of claim59, wherein the pharmaceutical composition inhibits cancer metastasis.61. The method of claim 59, wherein the pharmaceutical compositioninhibits growth of cancer cells that express MUC1*.
 62. A method fordiagnosing cancer or cancer metastasis comprising contacting cells of anindividual suspected of having cancer or a cancer metastasis with themonoclonal antibody of claim
 50. 63. The method of claim 62, wherein thecells are cells of a tissue or tissue sample.
 64. The method of claim62, wherein the cells are in vitro.
 65. A cell comprising a nucleic acidencoding the monoclonal antibody of claim 50.